CN1390253A - High order nucleic acid based structures - Google Patents

High order nucleic acid based structures Download PDF

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CN1390253A
CN1390253A CN00815433A CN00815433A CN1390253A CN 1390253 A CN1390253 A CN 1390253A CN 00815433 A CN00815433 A CN 00815433A CN 00815433 A CN00815433 A CN 00815433A CN 1390253 A CN1390253 A CN 1390253A
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nucleic acid
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polynucleotide
polynucleotide structure
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F·J·卡尔
G·卡特
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Merck Patent GmbH
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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

High order nucleic acid based structures
The present invention relates to and other molecular entity, the especially nucleic acid based molecular structure of entity bonded except that nucleic acid itself.The invention particularly relates to by combine and influence thus the nucleic acid based molecular structure with pharmaceutical active of morbid state with the specific molecular target.The present invention also relates to have the nucleic acid based molecular structure of diagnosis effectiveness.
Being starved of provides and can specificity change the activity of specified protein or the composition of the material that adjusting specific gene product is expressed.Special hope has the molecule that can form the specificity binding interactions with other molecule, can show this quasi-molecule of specificity bonded especially in vivo in the environment.
At these demands, developed several different methods, make it possible to prepare all kinds of molecular libraries of therefrom selecting such composition of matter.A relevant example is the very sensitive binding specificity that sees antibody molecule, and existing at present several effective technologies are used to develop monoclonal antibody and recombinant derivative thereof.A large amount of medicines and many diagnosis and research tool are provided.All such goods all are protein molecules, therefore only can utilize biosystem to produce.On the other hand, produced complete synthesis binding molecule.These binding molecules are the molecules in different libraries that are selected from the similar or metagon of identical chemical classes, and generally adopt screening system to select, and obtain surrogate or its active surrogate in some aspect of required treatment target.Huge small molecules chemistry library screening is the classical pathway of the conventional small-molecule drug of exploitation always, and synthetic at present peptide library and synthetic nucleic acid molecule library also are used for screening potential effective medicine.
For nucleic acid library, the general technology means are to utilize the single stranded RNA or the dna molecular of known unit length.With target molecule bonded basis not before configuration, but may depend on promote with other molecular entity bonded DNA (or RNA) molecule itself in secondary structure formation (1992 Nature such as Bock L.C. 355: 564-566; Kubrik, 1994 NucleicAcids Res. such as M.F. 22: 2619-2626).Do not attempt preparation treatment in the past and diagnose effectiveness, the nucleic acid molecule of (or senior) node configuration presequence section that contains secondary, purpose of the present invention that Here it is.
The present invention relates to the new purposes of novel high order nucleic acid structure and this structure.
The high order nucleic acid structure of several types is that the prior art field is known.A kind of such nucleic acid is called suitable (aptamer), and different with molecule of the present invention with regard to its size, preparation and topological complexity.The method that relates to from huge random library external evolution or selection be applicable to the fit and DNA of exploitation RNA fit both.Can promote enzyme process for example the active RNA molecule of polynucleotide kinase select circulation evolve (Lorsch J.R. and Szostak J.W.1994 Nature by repeatability 371: 31-36), and filtered out and can suppress human phosphatidase A by high degree of specificity 2Short single strand dna (1994 NucleicAcids Res. such as Bennett C.F 22: 3202-3209).Other investigator stand-alone development go out can in conjunction with and suppress the fit or DNA type is fit (1992 Nature such as Bock L.C. of the RNA type of some function of human thrombin 355: 564-566; Kubrik, 1994 Nucleic Acids Res. such as M.F. 22: 2619-2626).
The high order nucleic acid structure of other type comprises " branched DNA " (Horn, T. and Urdea, M.S.1989, Nucleic-Acids-Res. 17: 6959-6967), thus with one or more zones (" probe ") of the dna molecular of complementary nucleic acid molecule hybridization itself can with other dna molecule hybridize so that the amount of amplification and described dna probe bonded DNA.Yet the mixture that Horn and Urdea (ibid) describe is a linear extension, thus the new hybrid nucleic acid molecule of design not with previous annealed molecular hybridization, form the branched DNA structure and hybridize with other recruit of input.In fact, design such mixture exactly and form branch as much as possible, so that the annealing point of more signal nucleic acid probes is provided.
Materials science and field of nanometer technology have adopted nucleic acid base pairing other geometry (Aliviatos, 1996 Nature 382:609-611 such as A.P. that characteristic provided; 2000 Nature such as Mao C. 407: 493-496; Yurke, 2000 Nature such as B. 406: 605-608).Existing about producing and analyze introduction (Chen J. etc. 1989, the J.Am.Chem.Soc. of the exquisite technological method of high order nucleic acid based structures (comprising multiple tetragon, cuboctahedron and the trilateral motif that connects into grid) 111: 6402-6407; Zhang etc. 1994, J.Am.Chem.Soc. 116: 1661-1669; No. the 5th, 278051, United States Patent (USP); United States Patent (USP) the 5th, 468, No. 851; United States Patent (USP) the 5th, 386, No. the 6th, 072,044, No. 020 and United States Patent (USP)).Described solid is to adopt to relate to the closing structure that alternative manner that Restriction Enzyme is connected with DNA assembles.Interchain cross connection that can be by producing rigid structure or obtain node in the fixing described structure of more flexible branch by intersecting the visibly different side chain of annealing.The geometry that prior art does not comprise not closed (promptly terminal the connection).Prior art does not comprise the geometry that comprises the modification of nucleic acids district, and does not comprise and how much nucleic acid constructs of other molecular entity bonded.Prior art does not comprise that application at random or half random nucleic acid structure library.
About the new purposes of high order nucleic acid structure, known employing " rudimentary " nucleic acid itself is known in the art as treatment and diagnosis molecule.Specifically, many have potential therapeutic activity and or the examples of the active nucleic acid molecule of actual therapeutic are arranged.These molecules work as the RNA molecule (" ribozyme ") that antisense molecule, triplex agent or conduct have endoribonuclease activity.In the outward appearance of all these molecules, the pattern of treatment nucleic acid belongs to by weakening or the protein expression conditioning agent of the mechanism of action of blocking protein translation.Under all these situations, the target binding specificity is the binding specificity of nucleic acid and nucleic acid.These features (translation adjusting, nucleic acid and nucleic acid combination) are different with pattern of the present invention.A special inventive features of the present invention is to use to have with target molecule to combine active high order nucleic acid structure.
Tested other " rudimentary " nucleic acid construct, especially fit that obtains as treatment and diagnosis molecule.Identify some other nucleic acid molecule, especially ribozyme because have enzymic activity, and had potential pharmacy value.For the geometry of closure, though having inferred its conduct for the 5th, 278, No. 051, United States Patent (USP) is used for the possible effectiveness of the solubilizing agent or the controlled release carrier of small molecules medicine, do not consider its pharmacy effectiveness.
A first aspect of the present invention relates to novel high order nucleic acid based structures, particularly opening geometry.In addition, the invention still further relates to the purposes of this class formation as medicine and/or diagnostic reagent.The present invention also relates to comprise the high order nucleic acid based structures of modified nucleotide.The present invention also relates to comprise the high order nucleic acid based structures in randomization or half randomization Nucleotide district.The present invention also relates to the high order nucleic acid based structures puted together with other molecular entity (for example protein).
Structure of the present invention is utilized the Watson-Crick base pairing rules in the engineering district of duplex structure.Single stranded nucleic acid molecule by the complementarity between base can with other single chain molecule annealing (hybridization).Although such base annealing of two kinds of single chain molecules produces the line style duplex molecule usually, can also produce other structure, for example have under the situation of inner base pair complementarity at a molecule, can produce hairpin loop; And have under the situation of mutual complementarity at two ends of each single chain molecule, then can produce ring texture.Utilize the design of single-chain nucleic acid sequence strategy, can design can with two or more other molecules annealed individual molecule simultaneously, if these other molecules so can also with comprise other molecule annealing that participates in the annealed molecule, then can form nucleic acid complexes.
The overall space structure and the topology of double chain DNA molecule are well-known.Double chain DNA molecule is quite flexible, and the dna molecular duplex can be taked along the different many conformations of rotation angle between the adjacent base pair of spiral.Naturally occurring single stranded nucleic acid molecule for example RNA adopts the suitableeest conformation in solution.Described conformation depends on that same intramolecule base pair interacts, and produces the rock steady structure of being made up of double-stranded stem and single-stranded loop thus.Described molecule will adopt the conformation of minimum energy, but and this lowest energy structure method of calculation of known RNA sequence predict (1989 Proc.Natl.Acad.Sci USA such as Jaeger J.A. 86: 7706-7710).Attempt the folding software of production forecast DNA, and obtained certain success (1995 Nucleic Acids Res. such as Nielsen D.A. 23: 2287-2291).Space structure comparative descriptions between nucleic acid molecule and the protein molecule, the structural arrangement difference highly significant between this two quasi-molecule, and support basic design of the present invention.Typical case's globular preteins for example molecular weight is the myohaemoglobin of 17kDa, and its longest dimension is 3nm.Bigger globular preteins for example molecular weight is the bovine serum albumin of 68kDa, and its longest dimension is that (Cohen C. is stated from Wolstenholme G.E.W. and O ' Connor M. (writing), Ciba FoundationSymposium, London, J﹠amp to 5nm; A Churchill, 1966).The diameter of double-stranded spiral is originally as 2nm, and base logarithm few (for example 100) DNA chain contour length is about 30nm.Therefore, though the density of DNA or any other nucleic acid molecule is more much lower than typical protein, but during the most common natural structure form duplex of the topology of dna molecular even its, dna molecular can easily cover the major part of almost any protein molecule exposed surface.When if the topology of especially common monofilament DNA also so changes, described DNA can occupy most of area of space in the mode that more is similar to the much higher protein molecule of density.The nucleic acid construct that known assembling is made up of many interconnection chains (every chain for lacking the nucleotide sequence section of (＜50) very much) can easily obtain the structure of overall dimension 10-500nm.A special purpose of the present invention provides such nucleic acid molecule preparation.
Architecture basics of the present invention is that preparation is because two or more nucleic acid molecule interact or because interior different DNA or the RNA secondary structure molecules that section interacts and forms that limit of each nucleic acid molecule.In the present invention, the information content of DNA or RNA is not as the proteic coding entity of expression treatment, neither be used as the blocking-up entity (antisense) of nucleic acid metabolism and genetic expression, but in order to instruct assembling specific three dimensional shape molecular structure.
The present invention includes the nucleic acid molecule, especially the synthetic dna molecular that form three-dimensional (on-plane surface) molecular structure by particular bases pairing in the molecule in its group.Say that exactly the design dna molecule makes it contain the sequence area (" structural domain ") that one or more could anneal, finally form the complex three-dimensional nucleic acid construct with other molecule in its group.In this scheme, approximate cube structure can form by 6 synthetic dna molecule self-annealings, each synthetic dna molecule contains 4 complementations (complimentarity) structural domain, thereby each molecule and 4 other interactions of molecules, and each molecule effectively plays a role, as six cubical each sides.Described structure is open (not covalence closed), flexible, especially also is embodied in to be fit to modify by adding other functional group or building stone.
In a kind of high order nucleic acid based structures of the present invention, the a plurality of nucleic acid molecule that provide each nucleic acid molecule to comprise the self-complementary sequence structural domain more than 2 or 2 make described nucleic acid molecule can the oneself folding and interact by particular bases pairing process and to form special three-dimensional molecular structure.Use for some, the chemical instability of known, unmodified dna molecular has been a sizable problem for the purposes that is used as such as medicine.At present existing several methods is avoided enzyme in order to the protection dna molecular and is attacked degraded.These methods generally comprise to use and modify phosphodiester backbone (methyl orthophosphoric acid, thiophosphatephosphorothioate, peptide nucleic acid(PNA)) or adopt phosphoramidite, thiophosphatephosphorothioate or phosphorodithioate linkage to add cap at 5 ' end or 3 ' end.A special purpose of the present invention is to adopt modification of nucleic acids or non-natural nucleic acid in described high order nucleic acid based structures.In addition, a feature that needs especially is to reach the increase flexibility by application mix chemistry and alternative non-natural nucleic acid main chain aspect binding specificity.
The nucleic acid subunit of high order nucleic acid structure of the present invention can be natural homotype or heterologous nucleic acids subunit, for example contains the DNA of RNA tract.RNA tract in the known dna spiral changes spiralization (Wang, A. etc., 1982 Nature in solution 299: 601-04).Limiting the nucleotide sequence section exists conformation diversity possibility aspect the binding specificity of change and target protein significant, this phenomenon is known in the art, the fit binding specificity of zymoplasm depends on short section (Griffin, 1993 Gene such as L. of senior tertiary structure 137: 25-31).In addition, non-natural phosphate backbone analogue can be used to enhanced stability, and can change the binding specificity with required target protein.(Latham, 1994Nucleic-Acids-Res. such as J.A. such as Latham 22: 2817-22) provide an example, in the random oligonucleotide storehouse, replaced thymidine by this with modified nucleotide 5-(1-pentynyl)-2 '-deoxyuridine.The present invention includes by the single-chain nucleic acid tract and be inserted in the molecule that the duplex structure tract in the described single-chain nucleic acid tract is formed, and synthetic other chimeric molecule that contains the different chemical substructure but adopt conventional base pairing rules to connect.This class formation also can make up and contain dna molecular and RNA molecule.
More senior molecular structure of the present invention can be assembled by single or multiple nucleic acid molecule according to any scheme of this area, and can comprise from very large the molecule for example various nucleic acids or the fragment of recombinant plasmid.After the autofolding of single linear DNA molecule (assembling automatically) or facilitation are folding, can make up described structure.Facilitation is folding can pass through protein entity (enzyme, for example ligase enzyme, topoisomerase, endonuclease, polysaccharase) or by mediating with non-albumen physical and chemical condition (pH, temperature, ion condition) interaction.On the other hand, perhaps comprehensive the above method can be by assembling described molecule with the interaction of molecules that is combined on the solid substrate, and perhaps being folded into more in whole or part assembling process, the DNA of higher structure spatially is defined or grappling.
A second aspect of the present invention provides and contains the partly nucleic acid molecule library of molecule at random miscellaneous, and wherein may have can be with ad hoc fashion and the interactional required topology of target molecule for some molecule.Comprise the nucleic acid molecule library on the one hand at of the present invention this, institute art nucleic acid has divided is characterized as the guiding skeleton that promotes assembling common structure subunit.In each subunit, mix the stochastic sequence section, make with regard to the activity during selective binding is measured library diversity and potential function maximization of utility.In a second aspect of the present invention, adopt the independent colony of n synthetic dna molecule (subunit) (group) to mix the embodiment in the library that forms.Aspect this, colony's number of nucleic acid subunit is big, and colony's number depend on the variable section internal memory of described subunit degree of randomization.Further make up subunit's diversity by position, change variable domains quantity (interleaving the fixed sequence program section) and any given varied texture length of field of change that changes described variable domains in other embodiments.Be preferably in and mix n independently colony of subunit in the anneal cycles, have the library of a plurality of nucleic acid constructs and each sequence polymorphism with generation.Obviously other embodiment can comprise the multiple value of a plurality of anneal cycles and Integer n.In this scheme, a special feature in described library is by appropriate design and arranges that complementary sequence section or homing sequence section can regulator subunit interphase interaction complexities.
A third aspect of the present invention is new purposes, especially pharmacy and the diagnostic uses of high order nucleic acid based structures.Aspect this, these structures can combine with certain target molecules, normally combine with albumen or protein target molecule.Comprise in embodiment preferred under the situation of single protein targets, imagined further embodiment, the protein complex that wherein said target is made up of a plurality of protein subunits (for example cell surface receptor), and that this albumen composition is done is as a whole by the molecule combination of first aspect present invention.Other embodiment of the third aspect comprises in conjunction with cellular targets or various cell by identifying in conjunction with the ability of first aspect present invention molecule.Further embodiment comprises and the target of cell, especially cell surface or combining of target mixture (comprising non-protein ingredient for example carbohydrate or lipid composition).Protein, carbohydrate and lipid entity and or its mixture can be disease specific entity or tissue or the normal composition of cell.Described target or target mixture comprise the host's property component in virion or viral compositions derived therefrom (for example capsid protein) or the capsid.Described target or target mixture can comprise metal ion or other inorganic chemistry material or chemical group in it is formed, and can be naturally occurring or introduce by handling with exogenous factor.The target acceptor for example can comprise IL-2 acceptor or other cytokine receptor for example IL-3 acceptor, M-CSF acceptor, GM-CSF acceptor and many other acceptors.Equally, such as by blocking-up IgE in conjunction with the IgE acceptor, and the IgE surface molecular of blocking crosslinked reactivation process simultaneously should be in demand surface molecular.Comprise that bunch other surface molecular of differentiation (T cell differentiation antigen) serial member is needed adjusting disease, the especially target of autoimmunization composition disease.
Design of the present invention makes to have application especially widely in treatment molecule field.Wish molecular structure excitement of the present invention or antagonism special receptor or enzyme process to produce the treatment effect, the shortcoming that does not have conventional pharmaceutical grade protein simultaneously is immunogenicity for example.Therefore, the present invention extends to relate to and is used for the treatment of or the method for preventing disease or illness, and described method comprises the described molecular structure that gives curee's significant quantity.The present invention also extend relate to this class formation in vivo with the application of in-vitro diagnosis aspect.
A fourth aspect of the present invention is included in the high order nucleic acid based structures that described structure includes modified nucleotide.Derivatize that especially need be by described library subunit and or by comprising that in its building-up process modified base (thiol choline base, biotinylation base, epsilon-amino derivatize base etc.) gives diversity, this can be independent of diversity that above-mentioned second aspect gives or exist simultaneously with described diversity.Therefore, obtain all have multifarious height diverse libraries on sequence composition level and the sequence length level.This class parameter can be fixed in limited range with being used for different targets because of different libraries.The high order nucleic acid structure also can contain the modified nucleotide that can give described structure specific needs characteristic except aforementioned stable or the feature in conjunction with regulating effect are provided.This extra required modification can of the present invention first or second aspect in implement, comprise the using hydrophobic tract, comprise psoralene or acridine group, connect the haptenic group biological example plain or with the different charged side chain group that connects of amino or carboxyl for example, to promote and the combining of certain target molecules.In a further preferred embodiment, this class group can be as the tie point of other molecule, and described other molecule is other nucleic acid molecule or such as the protein of antibody or enzyme for example.
A required feature of molecule of the present invention is all to have high stability in vitro and in vivo.The chemical constitution of described nucleic acid construct is not only highly influential, and the shearing infringement that the physics size of described molecule need control in solution minimizes, and function maximization of utility in vivo.For this reason, the present invention is preferably common little (＜80mer) the multichain nucleic acid construct that is built into of subunit.On the other hand, may need to adopt by than the large subunit (＞structure 80mer) formed, and belong to category of the present invention equally.
A fifth aspect of the present invention comprises the high order nucleic acid based structures that is connected with other molecular entity.Specifically, this aspect is included in the nucleic acid that one or more specific sites are connected with one or more specific sites on another molecular entity, and the modified nucleotide of fourth aspect present invention promotes to be connected with the specificity of described nucleic acid.This aspect particularly including with pharmaceutically or the high order nucleic acid based structures that the associated molecule entity is connected on the diagnostics, thereby described nucleic acid combines with disease-related specific molecular target, described then link molecule entity is used for opposing or detect described disease.
Pharmaceutically for example liposome, live microorganism or attenuated microorganisms, photolytic activity part and other are induced the molecular entity of vaccine effect to Xiang Guan entity Fc part, other antibody related entities, toxin, enzyme, medicine and prodrug, receptor stimulant or antagonist, acceptor molecule itself (especially ligand binding domains), radio isotope, the pharmaceutical active nucleic acid, the drug delivery carrier that comprise cytokine, antibody.Part, fluorescence dye, enzyme and the signal transport vehicle that related entities on the diagnostics for example produces chemiluminescence signal particularly including radio isotope, photolytic activity part be microballon for example.
Briefly say, the present invention includes following purpose:
A kind of three-dimensional polynucleotide structure, described structure are matched interactional a plurality of chain that interconnects by nucleic acid molecule or its section by the particular bases of two or more molecules and are formed, and it is characterized in that described structure is not covalence closed.
A kind of corresponding polynucleotide structure is characterized in that described structure is by two or more nucleic acid molecule chain formation.
A kind of corresponding polynucleotide structure is characterized in that described structure is by three or three above nucleic acid molecule chain formation.
A kind of corresponding polynucleotide structure is characterized in that described structure is cubic form or is essentially cubic form.
A kind of corresponding polynucleotide structure, wherein said cube structure are by 6 nucleic acid molecule chain formation, and wherein the effect of every molecular chain is as six cubical each sides.
A kind of corresponding polynucleotide structure, wherein each nucleotide sequence comprise two or more can with other molecule annealed structural domain in its group.
A kind of corresponding polynucleotide structure, wherein each nucleotide sequence comprises four structural domains.
A kind of corresponding polynucleotide structure, wherein said structure comprise by the single-chain nucleic acid tract and are inserted in the nucleic acid molecule that the duplex structure tract in the described single-chain nucleic acid tract is formed.
A kind of accordingly according to each polynucleotide structure among the claim 1-8, wherein every nucleic acid chains less than 80 Nucleotide, be preferably less than 50 Nucleotide.
A kind of corresponding polynucleotide structure, wherein said structure has assembling shown in Figure 1 (A1+B1+C1)+(A2+B2+C2).
A kind of structure of polynucleotide as defined above, wherein said structure contains the subunit that is made up of variable randomized sequence section so that obtain can with target molecule interactional partly molecule or its section at random.
A kind of three-dimensional polynucleotide structure, described structure contains nucleic acid molecule or its section and matches interactional a plurality of subunit that chain is formed that interconnects by the particular bases of two or more molecules, wherein said structure is covalence closed, and contain the subunit that forms by variable randomized sequence section, so as to obtain can with target molecule interactional partly molecule or its section at random.
A kind of structure of polynucleotide as defined above, wherein said sequence is formed and sequence length is variable.
A kind of corresponding polynucleotide structure, one or more modifications that wherein said variable sequence is formed by described sequence kernel thuja acid obtain.
A kind of structure of polynucleotide as defined above, wherein said structure contain a plurality of can with other molecule or solid substrate in conjunction with or the nucleic acid site or the group that are connected.
A kind of corresponding polynucleotide structure, wherein said other molecule are protein, enzyme, lipoprotein, glycosylated protein, immunoglobulin (Ig) or its fragment.
A kind of corresponding polynucleotide structure, wherein said other molecule is a nucleic acid.
A kind of corresponding polynucleotide structure, wherein said molecule are pharmaceutically effective molecules.
A kind of medicinal compositions, described composition comprise a kind of as defined above, the polynucleotide structure in these claims and optional suitable carriers, vehicle and thinner and/or other compounds effective pharmaceutically.
A kind of corresponding polynucleotide structure is as the purposes of diagnostic reagent.
A kind of purposes of the structure of polynucleotide as defined above is used to provide the multifarious library of the specific randomization topology of influence, so that obtain different functions and/or activity.
The accompanying drawing summary Fig. 1
Diagram is called A by six respectively 1, A 2, B 1, B 2, C 1And C 2Single chain molecule be assembled into open cube nucleic acid construct step by step.Assemble by the conventional antiparallel base pairing between the nucleic acid molecule.Shown molecule B 1And C 1And B 2And C 2Between the dimerization intermediate that forms.Shown molecule A 1, B 1And C 1And A 2, B 2And C 2Between the tripolymer structure that forms.The assembling of cube structure partly connects by described two tripolymers to be finished, and is expressed as molecule (A 1+ B 1+ C 1)+(A 2+ B 2+ C 2). Fig. 2
The sequence of oligonucleotide subunit I L2R-1 and IL2R-2 comprises to have with the IL-2 acceptor and combines active dna structure. Fig. 3
The sequence of TB-R1 of oligonucleotide subunit and TB-R2 comprises to have with human thrombin and combines active dna structure.
Embodiment
By following examples explanation the present invention, but should not think that embodiment limits the present invention in any scope.
Embodiment 1 adopts the dna structure that is selected from the dna structure library to suppress the method for IL2 dependent cell system.
Synthetic two synthetic dna molecule libraries, each library comprises a randomized sequence district.
Library A comprises following structural molecule:
5’AGTCCCAAGCTGGCT(N) 13CTCCATCGTGAAGTCAGCCAGCTTTGGACT
Library B comprises following structural molecule:
5’GACTTCACGATGGAGGTCAGAATGTGAATA(N) 10TATTCACATTCTGAC
These sequences Design are in order to promote intersecting annealing, and representative will be by mixing and intersect the subunit in the structure library that form of annealing according to the present invention program's different subunit.
Purifying reaching maximum stable when having serum factor, and is carried out by HPLC in synthetic oligonucleotide (subunit) library with phosphorothioate bond.Purification of oligonucleotides derives from GenoSys Biotechnologies (Cambridge, Britain).Adopt single round-robin intersection annealing assembling dna structure library.Library A of subunit and B are carried out sex change, mixing and annealing when temperature is 37 ℃ in the solution of 50mM Tris pH7.4,100mM NaCl, 5mM EDTA.With etc. volumetric molar concentration (1 M) carry out the mixing of library A of subunit and B.In other experiment, adopt different mol ratios to mix.Prove conclusively the assembling of described subunit by gel electrophoresis.
In the screening DNA structure library can with IL-2 acceptor (IL2R) extracellular domain bonded structure.This screening is adopted according to the method for having announced (Meidel, 1988 Biochem.Biophys.Res.Commun.. such as M.C. 154: 372-379; Meidel, M.C. etc. 1989, J.Biol.Chem. 264: 21097-21105) solubility of preparation reorganization IL2R carries out.Employing supplier (Bangs Labs, Fishers, IN, USA) Jian Yi scheme makes reorganization IL2R and surface active magnetic bead covalent attachment, and the IL2R-magnetic bead is as the affinity surface, to select integrated structure from described dna structure library.The IL2R-magnetic bead is reacted with described library comprising under the various experiment conditions that have chaotropic salt in the control reaction.Described library (DNA) concentration is about 100nmol in aforesaid annealing solution.After carrying out thorough cycles of washing with the solution of 75mM Tris.HCl, 200mM NaCl, 0.5% n-octyl glucoside pH8.0, directly reclaim binding molecule from described magnetic bead by polymerase chain reaction (PCR).Adopt standard reagent system and condition, (5 '-AGTCCCAAGCTGGCT) carries out described PCR to reclaim library A component to use primer PRA1.In independent reaction, primer PRB1 (5 ' GACTTCACGATGGAG) and PRB2 (5 ' GTCAGAATGTGAATA) are used for reclaiming library B component.Adopt the standard reagent system and method, described PCR product is cloned and checked order.
Reclaimed many sequences, and be accredited as and be derived from library A of subunit and the library B of subunit.Adopt the synthetic wherein pair of sequences of foregoing thiophosphatephosphorothioate chemistry.Purifying as discussed previously also assembles oligonucleotide IL2-R1 and IL2-R2 (sequence shown in Figure 2), uses it for the raji cell assay Raji of IL-2 antagonistic action then.
TALL-104 (ATCC#CRL-11386) is that a kind of human T cell leukemia cell is.Described cell is grown in suspension culture, its suitableeest growth needs IL-2.Described cell can be grown for some time during no IL-2, but its growth is obviously slowed down.Allow cell contain 50-100u/ml recombinant human il-2 (Life Technologies, Paisley, Britain) and in the Iscoves of the additional heat-killed foetal calf serum of 10% (v/v) the improvement Dulbeccos substratum (Life Technologies, Paisley, Britain) grow.At 8-10%CO 2Environment under culturing cell.With medium preparation annealing IL2-R1/IL2-R2 DNA prepared product that contains IL-2 and the contrast DNA dilution of sample liquid that contains the stochastic sequence of identical appearance length.Utilization does not have the parallel dilution series of medium preparation of IL-2.The dilution series concentration range is 50 M DNA to 390nM DNA.In 96 hole microtiter plates, adopt the incomplete fusion TALL-104 cell of plating the day before yesterday to measure.By centrifugal collecting cell,, add the substratum that contains DNA then and handled 48 hours with warm (37 ℃) phosphate-buffered salt water washing in advance.Processing is carried out with four duplications.When 48 hours finish, with colorimetric estimation, tetrazotized zole compound that employing can commerciality obtains and the specification sheets assessment of proliferation that provides according to supplier (Promega, Southampton, Britain).Microtiter plate is at 540nm place reading.
The result shows that under the condition of each synthetic oligonucleotide IL2-R1 and IL2-R2 non-activity, described annealed dna preparation suppresses the growth of TALL-104 clone.
Embodiment 2 selects the method with human thrombin bonded dna structure.
With the library of describing among the embodiment 1 select can with human thrombin bonded dna structure.According to embodiment 1, the magnetic bead bonded human thrombin preparation (Sigma, Poole, Britain) of use and surface active screens described library.Make zymoplasm-magnetic bead and the reaction of described dna structure library according to embodiment 1, just the washing after the combination is at 20mM Tris acetate, pH7.4,140mMNaCl, 5mM KCl, 1mM MgCl 2Solution in carry out.Adopt reaction and the primer sets used,, directly from described magnetic bead, reclaim binding molecule by PCR as embodiment 1.Adopt the standard reagent system and method, described PCR product is cloned and checked order.
Reclaimed many sequences, and be accredited as and be derived from library A of subunit and the library B of subunit.Adopt the synthetic wherein pair of sequences of foregoing thiophosphatephosphorothioate chemistry.Purifying also assembles oligonucleotide TB-R1 and TB-R2 (sequence shown in Figure 3).The TB-R1/TB-R2 mixture is used for the blood coagulation EIA.Adopt fiber meter (fibrometer) in 37 ℃ and from adult's determination of plasma setting time of healthy donors prepared fresh.Adopt the setting time zymoplasm typical curve that mapping is drawn to concentration of thrombin, measure zymoplasm and suppress degree.In described mensuration, three logarithmic dna structures are measured setting time.
The result shows, when having described TB-R1/TB-R2 DNA mixture, blood coagulation activity is suppressed.
Embodiment 3 selects the method with recombinant soluble CD4 bonded dna structure.
With the library of describing among the embodiment 1 select can with recombinant soluble CD4 (rsCD4) preparation bonded dna structure.As discussed previously, adopt the described dna structure of CD4 preparation (BioDesign, Saco, ME, the U.S.) the screening library that is fixed on the activation magnetic bead.Library screening, washing and PCR select all as described in the embodiment 2.It is right that synthetic and assembling is derived from the single oligonucleotide in A subunit library and B subunit library.In enzyme-linked immunosorbent assay (ELISA), with the combination of the anti-CD4 mono-clonal of described STRUCTURE DEPRESSION RPAT4 (Serotech, Abingdon, Britain).
Be cushioned in the liquid (0.05M carbonate-bicarbonate buffer pH9.0) at bag, 96 hole elisa plates spent the night in 4 ℃ of bags with 0.2mg/ml rsCD4 solution.Thoroughly washing each plate with TBS-T (tris buffer saline pH8.0.05% (v/v) Tween 20), is that 100 Ms described plate dilute (1: 2) from initial concentration with dna structure to be measured and contrast dna structure in TBS.Each plate, is washed with TBS after 40 minutes in 37 ℃ of insulations.The 100ng/ml preparation of antibody RPAT4 in PBS added in the described plate, in 37 ℃ of insulations 40 minutes.After the wash plate, adopt the sheep anti mouse preparation (Sigma, Poole, Britain) and the chromogenic substrate Sigma Fast OPD (Sigma, Poole, Britain) of alkali phosphatase enzyme mark, detect bonded RPAT4.In some is measured, described DNA and RPAT4 monoclonal antibody are hatched altogether.The utilization card reader is read colored intensity, and relatively treats the signal of gaging hole and control wells.The result shows, when having described dna structure, significantly suppresses combining of RPAT4 and rsCD4.

Claims (22)

1. three-dimensional polynucleotide structure, this nucleic acid construct is interacted by the particular bases pairing by two or more molecules of nucleic acid molecule or its section and interconnective a plurality of nucleic acid molecule chain is formed, and it is characterized in that described structure is not covalence closed.
2. the polynucleotide structure of claim 1 is characterized in that described structure is by two or more nucleic acid molecule chain formation.
3. the polynucleotide structure of claim 2 is characterized in that described structure is by three or three above nucleic acid molecule chain formation.
4. each polynucleotide structure among the claim 1-3 is characterized in that described structure is cubes or is essentially cubic form.
5. the polynucleotide structure of claim 3, wherein said cube structure is by six nucleic acid molecule chain formation, and wherein the effect of every molecular chain is as six cubical each sides.
6. each polynucleotide structure among the claim 1-5, wherein each nucleotide sequence comprise two or more can with other molecule annealed structural domain in its group.
7. the polynucleotide structure of claim 6, wherein each nucleotide sequence comprises four structural domains.
8. each polynucleotide structure among the claim 1-7, wherein said structure comprise by the single-chain nucleic acid tract and are inserted in the nucleic acid molecule that the duplex structure tract in the described single-chain nucleic acid tract is formed.
9. each polynucleotide structure among the claim 1-8, wherein every nucleic acid chains is less than 80 Nucleotide.
10. the polynucleotide structure of claim 9, wherein every nucleic acid chains is less than 50 Nucleotide.
11. each polynucleotide structure among the claim 1-10, being assembled into of wherein said structure (A1+B1+C1)+(A2+B2+C2) shown in Figure 1.
12. each polynucleotide structure among the claim 1-11, wherein said structure contains the subunit that is made up of variable stochastic sequence section, so as to obtain can with target molecule interactional partly molecule or its section at random.
13. three-dimensional polynucleotide structure, this nucleic acid construct is interacted by the particular bases pairing by two or more molecules of nucleic acid molecule or its section and interconnective a plurality of nucleic acid molecule chain is formed, wherein said structure is covalence closed structure, and contain the subunit that forms by variable stochastic sequence section, so as to obtain can with target molecule interactional partly molecule or its section at random.
14. each polynucleotide structure among the claim 1-13, wherein said sequence is formed and sequence length is variable.
15. forming, the polynucleotide structure of claim 14, wherein said variable sequence realize by described sequence kernel thuja acid is carried out one or more modifications.
16. each polynucleotide structure among the claim 1-15, wherein said structure contain a plurality of can with other molecule or solid substrate in conjunction with or the nucleic acid site or the group that are connected.
17. the polynucleotide structure of claim 16, wherein said other molecule are protein, enzyme, lipoprotein, glycosylated protein, immunoglobulin (Ig) or its fragment.
18. the polynucleotide structure of claim 16, wherein said other molecule is a nucleic acid.
19. each polynucleotide structure among the claim 16-18, wherein said molecule are the effective molecules of pharmacy.
20. a medicinal compositions, said composition comprise polynucleotide structure and optional suitable carriers, vehicle, thinner and/or other pharmacy active compound of claim 19.
21. the purposes of each polynucleotide structure among the claim 1-18, this purposes are that described polynucleotide structure is as diagnostic reagent.
Influence the multifarious library of specificity randomization topology 22. the purposes of the polynucleotide structure of claim 12 or 13, this purposes are described polynucleotide structure to be used to provide, so that obtain different functional and/or active.
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