EP0151631A1 - Jonctions ramifiees d'acide nucleique ayant une mobilite de migration definie de maniere precise - Google Patents

Jonctions ramifiees d'acide nucleique ayant une mobilite de migration definie de maniere precise

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Publication number
EP0151631A1
EP0151631A1 EP19840903127 EP84903127A EP0151631A1 EP 0151631 A1 EP0151631 A1 EP 0151631A1 EP 19840903127 EP19840903127 EP 19840903127 EP 84903127 A EP84903127 A EP 84903127A EP 0151631 A1 EP0151631 A1 EP 0151631A1
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European Patent Office
Prior art keywords
composition
junction
nucleic acid
double
branched
Prior art date
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EP19840903127
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German (de)
English (en)
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Nadrian Charles Seeman
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Research Foundation of the State University of New York
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Research Foundation of the State University of New York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • This invention relates to a new composition of matter comprising a polynucleotide containing at least one immobile or semi-mobile branched junction, and to the method of making it.
  • oligomeric nucleic acids form mobile linear duplexes stabilized by hydrogen bonds between bases.
  • Polymeric nucleic acids occasionally branch to form junction structures in nature, as shown in Figs. 1 and 1A of the drawings. These branched duplexes are unstable, and resolve to form two independent duplexes.
  • the pre-eminent structural characteristic of stable double helical nucleic acids in nature is that the positions of all atoms in the molecule bear a well-defined relationship to a linear (although not necessar ily straight) axis which exhibits no junctions (branch points). Nevertheless, conformational variability (Kim, S.H.,
  • junction structures are involved as intermediates in single-strand-displacement recombination and as transcriptional intermediates, such as that shown in Fig. 1A.
  • Fig. 1A transcriptional intermediates
  • the strands there shown are unlikely to form junction structures in preference to double helices; if they did occasionally combine to form such structures, the process of branch point migration, shown in Fig. 1 will result in the rapid resolution of the junction structures into double helices (Thompson, B.J. Camien, M.N., and Warner, R.C., Proc. Nat. Acad. Sci. (USA) 73, 2299-2303 (1976)).
  • the synthetic oligomeric nucleic acids of this invention are so constructed as to form migrationally immobile or semi-mobile branched junctions. These new stable oligomeric structures, stabilized by maximizing Watson-Crick base pairing, minimize the sequence symmetry found in their unstable analogs in living systems.
  • the synthetic oligonucleotide sequences of the compositions of this invention contain migrationally immobile and/or semi-mobile junction structures. In a semi-mobile junction a limited degree of configurational degeneracy is introduced into the system. These DNA junctions represent nexi, from which 3 to 8 double helices may emanate.
  • Each junction of these compositions may be treated as a macromolecular "valence cluster" containing individual clusters which may be linked together directly, or with segments of linear DNA interspersed between them.
  • the covalently linked compositions can be formed with a high degree of specificity, using the state-of-the-art sticky-ended ligation techniques currently employed in genetic engineering.
  • the covalently joined three-dimensional new networks of nucleic acids containing immobile or semi-mobile junctions are periodic in connectivity and may also be periodic in space, thereby generating quasicrystalline arrays of matter.
  • compositions are described herein in terms of DNA, but they also include RNA, RNA-DNA-Hybrids or nucleic acids in which the backbones or bases have been modified, but not so as to affect their pairing capabilities, via Watson-Crick or some other form of association.
  • the junctions are then formed simply by dissolving the selected sequences in the desired proportions in a suitable liquid solvent, preferably an aqueous buffer, at a temperature from about 0° to about 60°C, usually at 20°-40°C, and at pH 6-9 and in the presence of a counterion.
  • Fig. 1 is a schematic representation of a portion of a polymeric nucleic acid containing an unstable, mobile junction as it occurs in nature;
  • Fig. 1A is a schematic representation of a replicational junction
  • Fig. 2 is a schematic representation of a synthetic oligomeric nucleic acid containing an immobile junction which is one embodiment of the invention
  • Fig. 3 is a view of a stained gel chromatogram sheet containing the composition of Fig. 2 in lane K
  • Fig. 4 is a view of a stained gel chromatogram sheet containing the composition of Fig. 2 in lanes A to E, inclusive
  • Fig. 5 is a graph showing ultraviolet absorption of the composition of Fig. 2, and of strands 1 and 2 and of strand 3, at varying temperatures;
  • Fig. 6 is a flow chart showing the steps for optimizing sequences of synthetic oligomeric nucleic acid strands for making compositions of the present invention
  • Fig. 7 illustrates an odometer analogy to an algorithm used in the optimizing procedure of Fig. 6
  • Fig. 8 is a schematic representation of a composition which is a second embodiment of the invention
  • Fig. 9 is a view of an autoradiogram of a gel chromatographic sheet containing the composition of Fig. 8 in lanes G and H;
  • Fig. 10 is a schematic representation of compositions of the invention containing immobile junctions of Rank 3 to 6;
  • Fig. 11 is a view of an autoradiogram of a gel chromatogram containing varying amounts of the composition of Fig. 8 in lanes B-E and in lanes G-J respectively;
  • Fig. 12 is a schematic representation of a composition according to the invention containing a plurality of immobile junctions interconnected to form a lattice or network;
  • Fig. 13 is a schematic representation of a composition according to the invention containing a semi-mobile junction which is capable of existing in only the two interchangeable or isomeric forms shown;
  • Fig. 14 is a schematic representation of a composition according to the invention containing a Rank 3 immobile junction and of a composition containing a plurality of such junctions interconnected to form a lattice or network;
  • Fig. 15A shows a portion of the NMR spectrum of the composition of Fig. 14;
  • Fig. 15B shows the corresponding portion of the. NMR spectrum of a single strand of the composition of Fig. 14.
  • the construction of new immobile junction compositions of this invention requires the ability to identify and select unique sets of sequences possessing conventional Watson-Crick base pairing patterns while at the same time minimizing sequence symmetry. Sequences containing long sequences of base pairs can be formed at higher temperatures, up to about 60°C or even higher. The probability of forming a desired junction is a function of the free energy of association of the individual strands involved.
  • each strand which is chosen to participate in the formation of an immobile or semi-mobile junction can be considered to be composed of a series of overlapping segments of a given criterion length, Nc.
  • Nc criterion length
  • each hexadecameric strand in the immobile junction shown in Figure 2 is a series of 13 overlapping segments of length 4.
  • Each of these segments is termed a "criton”, while the complement to a criton, i.e. the sequence of bases with which it pairs, is termed its "anti-criton”.
  • Watson-Crick pairing arrangements which compete with the desired pairing must be considered from a thermodynamic point of view for lengths less than Nc.
  • the same base pair can only abut the junction twice. If it is present twice, those two occurrences must be on adjacent arms.
  • the first three rules ensure immobility through lack of homology, except for Nc-1 base pairs on each arm belonging to the criton nearest the junction.
  • the fourth restriction eliminates migratory possibilities for these bases; it should be applied sequentially to each base pair in the criton, and perhaps beyond.
  • the fourth rule also limits the maximum rank of junctions: Since there are only four base pairs, A-T, T-A, G-C, C-G, and since each pair can only appear twice, junctions of rank greater than eight are not possible with the conventional bases.
  • Nc is a number to be minimized, since this minimizes the strengths of competing interaction by shortening the lengths involved.
  • the most likely reason for increasing Nc is that the desired junction cannot be generated by the 4 Nc critons available for a given value of Nc.
  • the constraints applied to the generation of the immobile junction in Figure 2 are incompatible with a value of Nc less than 4.
  • a junction in which the limited amount of mobility is non-zero i.e., a semi-mobile junction, such as the one shown in
  • a FORTRAN computer program is described hereafter which generates sequences that fulfill these criteria for junctions of any design by a rapid algorithm.
  • the program also ranks generated sequences on the basis of pairing fidelity relative to competing interactions at lengths shorter than Nc.
  • the details of the algorithm and the way in which free energy considerations are taken into account for competing Watson-Crick pairing interactions (at lengths shorter than Nc) will be discussed elsewhere in this description.
  • the specificity of double helical Watson-Crick base pairing is then utilized to link up two different pieces of DNA possessing complementary sticky ends to produce a composition containing two or more immobile or semi-mobile junctions.
  • 2- and 3-dimensional networks which are possible, some are of course periodic in their connectivity. Such networks can also be periodic in space and can then comprise unique crystalline macropolymers of a size suitable for diffraction analysis using x-rays, and perhaps neutrons.
  • An example of such a 2-dimensional network is shown in Figure 12, illustrating formation of a twodimensional lattice from an immobile junction with sticky ends. A is a sticky end and A' is complementary to it. A similar relationship exists between B and B'.
  • the architecture of a junction requires the specifications of both covalent connectivity and base pairing relationships. Because of the complementary nature of the Watson-Crick double helices which constitute the junction structure, only half of the bases must be treated as independent variables; those bases complementary to them are treated as dependent variables. Bases are encoded as numbers on to base 4. In the case of semi-mobile junctions only one out of four of the mobile bases is independent. With a computer, new sequences may be generated simply by the process of counting in base 4. If all of the arms of a junction have the same length, it is possible to fix one base at the numerical level, thereby decreasing the number of independent variables by one. The independent bases may be ordered by the rapidity of the rates of change of the digits representing their identities within the program.
  • order is meant an inverse measure of the rate at which the digit representing the base is incremented.
  • the lowest ordered base will be changed on every pass, the next lowest ordered base will change on every fourth pass, the next lowest ordered base on every sixteenth pass, and so on.
  • the critons themselves may be ordered according to the lowest ordered base within the criton. The critons are then tested for adherence to the rules sequentially, from highest to lowest order. Thus, if a given criton violates one of the rules, the base corresponding to the order of that criton is advanced, rather than the base of the lowest order.
  • Step 6 The nine logical steps in this procedure are indicated schematically in Fig. 6.
  • the two steps in double boxes must clearly be done by the programmer while the other steps are done automatically by the programs.
  • the covalent connectivity and desired base pairing are selected by the programmer. Specific constraints can also be introduced at this stage.
  • the critons are ranked by the order of the most rapidly changing base which they contain. After that, an initial numerical sequence must be assigned. (Step 3) This numerical sequence is tested against the junction rules (Step 4) and if it fails, a new sequence is generated by the fast algorithm. If the numerical sequence obeys the rules, its base permutations are the tested, (step 5) against investigator selected constraints.
  • Step 6 If any of the eight sequences implied by the numerical procedures are acceptable, their fidelities are calculated. (Step 6), and if these are acceptably high, melting curves are calculated and plotted, (step 7). New sequences are then generated (step 8) and tested iteratively. until all possibilities have been exhausted. The programmer may then evaluate the alternatives presented by the programs. Once the strand sequences fulfill the uniqueness and nonmobility rules, thermodynamic criteria-are applied to all sequences of length less than Nc.
  • the first question to consider is the pairing fidelity: Is the desired base pairing configuration the most probable configuration in which these particular sequences are to be found in solution? If so, what is its probability relative to other pairing configurations?
  • K AB ⁇ K 1 K 2 K 3 ... K N-1 , (1)
  • N is the chain length of complementary sequences between chains A and B
  • ⁇ is the nucleation constant
  • thermodynamic data are available from which primitive sets of K i, s can be created, together with rough values of the ⁇ H° i, s . Despite the uncertainties, these data make it possible to estimate the relative ⁇ ontr ibutions of different sequences with reasonable accuracy, particularly if appropriately scaled values from oligoribonucleotides are used.
  • the information contained in the estimated equilibrium constants for pairing specific sequences can be used to predict approximate thermal transition profiles for junctions.
  • enthalpy values, ⁇ H i ° corresponding to the equilibrium constants K i used to assess fidelity, are required. These are considerably less certain for oligodeoxynucleotides than for oligoribonucleotides, but nonetheless reasonable estimates are available, and missing values can be filled in by scaling the corresponding RNA data, as described.
  • the value of K AB and the starting concentrations of the two species uniquely characterizes the equilibrium; for starting concentrations, C A and C B (moles per liter).
  • C AB can be calculated. This can be done at any temperature if the ⁇ H i ° for each K i is known.
  • K ABCD The value of K ABCD is estimated as :
  • K ABCD ⁇ -1 (( ⁇ R k BC k DA )+k BC + k DA ).
  • oligodeoxynucleotide strands synthesized from appropriately blocked nucleotides or dinucleotides by standard chemical procedures are placed in a glass or plastic vessel at room temperature in a solvent containing (1) a buffer system capable of regulating pH between pH6 and pH9, so as to favor Watson-Crick base-base hydrogen bonding and (2) a source of counterions in order to reduce the electrostatic repulsion among the strands as they form a ternary, quaternary (or higher) junction or complex.
  • Suitable buffers include phosphate, cacodylate, tris, etc. at concentrations from 0.001 to 1.0 M approximately.
  • the pH can be regulated by direct titration to yield an end-point in the above range; the pH so obtained is less stable to temperature and electrophoresis.
  • Satisfactory counterions have been found experimentally to be (a) 1-2 molar NaCl, KCl, CsCl or any non-destabilizing monovalent neutral salt including Na 2 SO 4 , K 2 SO 4 , Cs 2 SO 4 , (b) 5-10 mM Mg 2 Cl, Ca-Cl or comparable divalent neutral salt or (c) 1-5 mM spermine, spermidine or other neutral alkyl diamines NH 3 +-R-NH 3 + or (d) combinations of the above agents. Lack of suitable counter ions destabilize the junctions selectively relative to simpler structures (dimers, e.g.).
  • hypochromicity of the strong ( ⁇ 260nm) ultraviolet absorbance of the bases (1) hypochromicity of the strong ( ⁇ 260nm) ultraviolet absorbance of the bases;
  • Example 1 The four blunt-ended oligomeric helical strands of DNA designated in Fig. 2 were selected by means of the rules and algorithm described above, then were synthesized by conventional phosphotriester technique on a commercially available synthesizer from appropriately blocked nucleotides or dinucleotides.
  • This sequence also contains no repeating GpG sequence longer than two, in order to restrict the possibility of GG non-Watson-Crick pairing as well.
  • These sequences were lyophilized, and equal weights of the four strands were then dissolved to form a 2 mM solution in an aqueous buffer (50 mM phosphate or tris pH 7; 10 mM MgCl 2 ) at room temperature, whereupon the immobile junction of Fig. 1 formed spontaneously.
  • the buffer solution was subjected to electrophoresis on polyacrylamide gel (10-15% acrylamide) along with solutions of other materials in separate lanes which provided mobility references.
  • chromatogram was stained with a conventional dye (stains-All, Kodak) which colors single as well as double stranded nucleic acids; the results were as shown in Fig. 3, in which lane K contained the desired composition having an immobile junction as shown in Fig. 2.
  • a conventional dye stains-All, Kodak
  • each of the other wells or lanes contained other materials to provide mobility references only.
  • Lanes a, b and 1 contained restriction digests of PhiX-174-RF-DNA; a is the Hinf I digest, b is the Hae III digest and 1 is the Hinc II digest. The lowest molecular weight fragments in these digests are: 42,48,66 and 82
  • Lanes c-f contained strands 1, 2, 3 and 4 respectively.
  • Lane g contained an equimolar mixture (based on extinction coefficients derived from dry weights) of strands 1, 2, and 3; lane h, strands 1, 2, and 4; lane i, strands 1, 3, and 4; and lane j, strands 2, 3, and 4.
  • Lane k contained an equimolar mix of strands 1, 2, 3 and 4.
  • Lane m to r contained equimolar mixtures of pairs of strands: m contains 1 and 2, n 3 and 4, o 1 and 4, p 2 and 3, q 1 and 3, and r 2 and 4. Lane k of Fig.
  • component (i) consisted of an equimolar mixture of strands, 1, 2 and 4 of Fig. 2 while component (ii) consisted of strand 3 alone, each in a different aliquot of the same buffer.
  • Lanes G to J contained 8 micrograms of free strands 4, 3, 2 and 1, respectively.
  • Lane F contained 6 micrograms of component (i).
  • Lanes A to E each contained 6 micrograms of (i), and in addition, 0.5 micrograms (E), 1.0 micrograms (D), 2.0 micrograms (C), 3.0 micrograms (B), and 4.0 micrograms (A) of component (ii).
  • the resulting chromatogram, after staining, is shown in Fig. 4.
  • Each of lanes A to E contained, in varying amount, a composition containing the immobile junction of Fig. 2.
  • ⁇ A 260 (A 260 (T)/A 260 (10°C)-1).
  • Typical ⁇ A 260 for high molecular weight DNA duplexes approaches 30%. Actual values depend on base composition, length and solvent.
  • Strand 3 alone exhibited a typical non-cooperative transition characteristic of nucleic acids in the absence of base pairing.
  • Example 2 The four dodecanucleotides depicted in Fig. 8 were designed by the use of the sequence symmetry minimization rules, supplemented by equilibrium distribution optimization as explained above. Note the lack of symmetry around the center so that migration is not possible. These sequences also contain no repeating GpG sequence longer than two, in order to minimize competition from this form of non-Watson-Crick pairing as well. These sequences were synthesized by conventional phosphotri ester techniques.
  • Unlabelled strand 1 was present, respectively, in quantities of 0 (A), 0.15 ⁇ g, (B) 0.3 ⁇ g. (C) 0.6 ⁇ g, (D) 1.2 ⁇ g (E). The counts in the top (junction) band are maximized in lane D, indicating 1:1:1:1 stoichiometry.
  • Lanes F-J represent a similar experiment in which the unlabelled strand was strand 4. Labelled strands 1, 2, and 3 were each present in 0.6 ⁇ g quantities in each of lanes F-J.
  • Unlabelled strand 4 was present in quantities of: 0 (F), 0.15 ⁇ g (G), 0.3 ⁇ g (H), 0.6 ⁇ g (I) and 1.2 ⁇ g (J). Again, the junction band saturates at 1:1:1:1 stoichiometry, corresponding to lane I.
  • Example 3 The three strands shown in the upper part of
  • Fig. 14 of the drawing were designed in accordance with the rules, and algorithm set forth above and were synthesized by conventional phosphotriester technique.
  • the vertical-appearing strand (containing 18 residues) was synthesized with blunt ends, while the ones at lower left (containing 20 residues) and at lower right (containing 22 residues) were synthesized each with a sticky end, as shown.
  • the strand at l nieower left (20 residues) was end-labelled by enzymatic kinase reaction with T4 polynucleotide kinase enzyme using gamma 32 P labelled ATP.
  • Each of the three strands was dissolved in the same specimen of buffer (tris pH7 with 16 mM MgCl 2 ) to a concentration of 1 mM to form a composition containing the immobile junction shown in the upper part of Fig. 14 and exposed to DNA ligase, a joining enzyme, for 24 hours at room temperature, using a large excess of the enzyme and ATP, its cofactor. Subsequently, the reaction was stopped, the mix was extracted with phenol, bubbled with ether to remove the phenol, and dried by lyophilization to form a solid. When subjected to electrophoresis in buffer, a specimen of this composition exhibited several different bands, of which those appearing as 4 and 6 multiplets of the single 20 residue band were very prominent.
  • composition When the composition was subjected to digestion with the exonuclease enzyme, exo III, which catalyzes hydrolysis of linear polynucleotides stepwise from their free ends, and the solution subsequently was electrophoresed on 10% polyacrylamide gel, only the two bands at the positions of the 4- and 6-multiplets remained, showing that the composition was in part in the form of the 6-multi ⁇ let hexagonal or circular unit structure or geometric network shown in the lower portion of Fig. 14, with the 20 mer str-and running along the inside of the hexagon being covalently joined; the remainder of the composition was in the form of a 4-multiplet square (or circular) unit structure or geometrical network.
  • the NMR spectrum (low field region of 600 MH ⁇ NMR spectrum of 2 mM solution) of another specimen of the lyophilized composition containing the three strands shown in Fig. 14 was determined, as shown in Fig. 15A.
  • the extremely broad line widths are characteristic of linear duplex DNA of chain lengths in excess of 260 base pairs, indicating hydrogen bonding of the sticky ends to form larger complexes containing a number of the immobile junctions in a geometric network.
  • the NMR spectrum of a single strand DNA composition shown in Fig. 15B, exhibits characteristically sharp lines.
  • Unlabelled specimens of the composition can be obtained by following the same procedure, using the autoradiogram as a guide to the location of the desired composition after electrophoresis.
  • Compositions having other similar unit structures can be made containing other immobile or semi-mobile junctions of rank 3 to 8 using strands having either a single sticky end or two sticky ends.
  • compositions containing mobile or semi-mobile nucleic acid junctions may be used as vertices of n-connected networks of nucleic acids. That is the fundamental aspect about this invention which gives it value. They can be used to make nucleic acid structures in the form of geometrical stick figures where the sticks correspond to double helical nucleic acids and the vertices are nucleic acid junctions.
  • the utility of being able to make the geometrical figures is that this allows one to do molecular engineering of this sort on the hundred to thousand A scale. This, in turn, allows one to make various kinds of intricate figures which may have utility as appropriate surfaces upon which to condense cognate mclecules such as the protein depicted in Figure 11.
  • a periodic structural network of this form would be an appropriate substrate for the investigation of any material, be it nucleic acid. something which naturally interacts with nucleic acid, or otherwise.
  • a reactive "hook” or “hooks” could be attached to one or more residues within the unit cell to covalently capture and identically orient the molecule of choice.
  • divalent antibodies covalently and/or non-covalently bound to nucleic acids or to cognate proteins could accomplish the same goal without using covalent reactions.
  • junction lattic can be used as a template for crystallizing and structurally characterizing materials that otherwise may not be readily crystallizable.
  • protein folding intermediates or messenger RNA molecules that perhaps cannot crystallize at all.
  • a great advantage of using junction lattices to look at such systems is that lattice forces would only affect the lattice molecules themselves, and not the molecules introduced into the lattice for structural study.
  • protein-folding intermediates and long RNA molecules with readily perturbed tertiary structures would be visible structurally without perturbing them with lattice forces.

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Abstract

Des jonctions ramifiées d'acide nucléique ayant une mobilité de migration définie de manière précise, comprenant de nouvelles compositions de matière contenant des jonctions ramifiées semi-mobiles et/ou immobiles d'acide nucléique d'où au moins trois doubles hélices émanent sont décrites. Ces compositions de matière peuvent comprendre des parties intégrales de réseaux périodiques ou autres ayant des dimensions moléculaires précises, permettant ainsi la constitution d'une architecture moléculaire exacte sur l'échelle de 100 à 1.000 Angstroem. Elles peuvent être produites par dissolution d'au moins trois polynucléotides ayant entre eux une symétrie séquentielle minimale.
EP19840903127 1983-08-03 1984-08-01 Jonctions ramifiees d'acide nucleique ayant une mobilite de migration definie de maniere precise Withdrawn EP0151631A1 (fr)

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US5124246A (en) * 1987-10-15 1992-06-23 Chiron Corporation Nucleic acid multimers and amplified nucleic acid hybridization assays using same
US5359100A (en) * 1987-10-15 1994-10-25 Chiron Corporation Bifunctional blocked phosphoramidites useful in making nucleic acid mutimers
US5424413A (en) * 1992-01-22 1995-06-13 Gen-Probe Incorporated Branched nucleic acid probes
US6072044A (en) * 1996-04-26 2000-06-06 New York University Nanoconstructions of geometrical objects and lattices from antiparallel nucleic acid double crossover molecules
BR0015484A (pt) * 1999-11-13 2002-07-02 Merck Patent Gmbh Estruturas à base de ácidos nucléicos de alta ordem
JP4061401B2 (ja) * 2002-03-07 2008-03-19 国立大学法人九州大学 Ｄｎａの自己組織化によるｄｎａナノケージ及びその製造方法、並びにそれを用いたｄｎａナノチューブ、分子キャリアー
CA2587010C (fr) 2004-11-08 2017-08-15 Vipergen Aps Sysnthese chimique guidee par acides nucleiques structurels
EP2241640B1 (fr) * 2008-01-22 2016-06-22 Hiroshima University Procédé et kit de dosage de protéine de liaison à l'acide nucléique

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