WO2003087188A1 - Polymers et copolymeres romp a modification oligonucleotidique - Google Patents

Polymers et copolymeres romp a modification oligonucleotidique Download PDF

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Publication number
WO2003087188A1
WO2003087188A1 PCT/US2002/012071 US0212071W WO03087188A1 WO 2003087188 A1 WO2003087188 A1 WO 2003087188A1 US 0212071 W US0212071 W US 0212071W WO 03087188 A1 WO03087188 A1 WO 03087188A1
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polymer
romp
oligonucleotides
bound
particles
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PCT/US2002/012071
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WO2003087188A8 (fr
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Chad A. Mirkin
Sonbinh T. Nguyen
Keith J. Watson
So-Jung Park
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Nanosphere, Inc.
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Priority claimed from US09/830,620 external-priority patent/US7115688B1/en
Application filed by Nanosphere, Inc. filed Critical Nanosphere, Inc.
Priority to AU2002367709A priority Critical patent/AU2002367709A1/en
Publication of WO2003087188A1 publication Critical patent/WO2003087188A1/fr
Publication of WO2003087188A8 publication Critical patent/WO2003087188A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • This application relates to ROMP polymers or co-polymers having oligonucleotides attached thereto, materials comprising the ROMP polymer or copolymer conjugates, and methods for preparing and using the same.
  • the present invention relates to ROMP polymers or co-polymers having oligonucleotides bound thereto, materials or structures comprising the same, and methods of preparing and using the same.
  • ROMP ring-opening metathesis polymerization
  • the applicants discovered that the covalent attachment of synthetic oligonucleotides to the backbone of a well-defined polymer structure derived from ring-opening metathesis polymerization (ROMP) reaction provides polymers that are useful in preparing novel materials.
  • Attempts to incorporate DNA into ROMP polymers via polymerization of monomers including DNA strands were unsuccessful.
  • monomers are polymerized using any suitable metathesis catalyst. If two or more different monomers are used, the polymerization may occur in a stepwise or simultaneous manner to produce block and random co-polymers.
  • Any suitable monomers may be used, however these monomers preferably include a reporter label and/or a functional group that would allow for post-polymerization modification of the preformed polymer template to attach oligonucleotides. Particularly prefened monomers are substituted norbornenes having reporter labels such as a UN tag or redox active fenocenes. Post-polymerization modification of the resulting ROMP polymer template with 2-cyanoethyl diisopropyl chlorophosphoramidite allows the polymer to be easily be modified with D ⁇ A using standard solid phase techniques. D ⁇ A-modifed ROMP polymers or copolymers with various redox potentials can be prepared which have full D ⁇ A recognition properties and electrochemical properties.
  • these polymers exhibit useful properties such as sharp melting transitions and high thermal stabilities.
  • the Examples below describe the ROMP polymerization of a norbornenyl-modified alcohol (2) substituted with a diphenylacetylene spacer as a UN- tag using a commercially available ruthenium-carbene catalyst.
  • the resultant ROMP homopolymer was then modified with the chlorophosphoramidite and coupled to predefined D ⁇ A molecules using solid phase technique.
  • the resulting D ⁇ A-modified ROMP polymers were characterized using UN-spectroscopy in combination with D ⁇ A hybridization studies.
  • Aggregate structures comprised of polymers with complementary strands led to the formation of extended hybridization networks which precipitate reversibly from aqueous solutions, demonstrating that multiple D ⁇ A strands are attached to each individual polymer.
  • D ⁇ A modified polymers were exposed to a solution containing 13 nm gold particles with complementary strands attached to their surface, three dimensional aggregates of particles were formed and characterized using UN-Nis spectroscopy and transmission electron microscopy.
  • block copolymers derived from 2 and norbornenyl-modified fenocenes can be synthesized and coupled to D ⁇ A using this strategy. The presence of the second block did not interfere with the recognition properties of the D ⁇ A and imparted electrochemical properties that are useful in detecting for the presence or absence of a target nucleic acid or oligonucleotide.
  • the present invention provides a ROMP polymer or co-polymer having oligonucleotides bound thereto.
  • the ROMP polymer may be a homopolymer.
  • the ROMP co-polymer comprises a ROMP block co-polymer or random co-polymer.
  • the ROMP block co-polymer includes multiblock co-polymer.
  • the oligonucleotides bound to the polymer may comprise a spacer portion and a recognition portion wherein the spacer portion is bound to the ROMP polymer, and the recognition portion having a sequence that is complementary to at least one portion of the sequence of another oligonucleotide.
  • the oligonucleotides comprise at least one type of recognition oligonucleotides, each type of recognition oligonucleotides comprising a spacer portion and a recognition portion wherein the spacer portion is attached to the ROMP polymer and the recognition portion has a sequence complementary to at least one portion of the sequence of another oligonucleotide.
  • the spacer portion may include from about 4 to about 30 nucleotides, preferably 10 nucleotides and most preferably about 4 nucleotides.
  • the monomer is preferably a cyclic mono-olefm such as substituted norbornene.
  • a suitable substituted norbornene include norbornenyl-modified alcohol such as monomer 2 which include a UN tag and a norbornenyl group modified with an electrochemical tag such as a norbornenyl-modified fenocene.
  • a ROMP polymer in another embodiment, comprises an oligonucleotide-modified product produced by the ROMP polymerization of monomer 2 to produce a homopolymer template and post-polymerization modification of the polymer template to attach oligonucleotides.
  • a ROMP co-polymer having oligonucleotides bound thereto is provided and that is produced by the process of (a) sequential block ROMP polymerization of monomer 2 and at least one or more different monomers to produce a ROMP co-polymer template; (b) post-polymerization modification of the template, followed by coupling of oligonucleotides to the modified template.
  • the one or more different monomers may include a substituted norbornene such as a norbornenyl group modified with an electrochemical tag, e.g a norbornenyl- substituted fenocene.
  • the present invention also provides materials or structures comprising a ROMP polymer or co-polymer having oligonucleotides bound thereto.
  • materials or structures are provided that comprise a first and second ROMP polymers or copolymers having oligonucleotides bound thereto, the oligonucleotides bound to the first ROMP polymer or co-polymer having a sequence that is complementary to the oligonucleotides bound to the second ROMP polymer or copolymer.
  • materials or structures are provided that are comprised of:
  • a connector for holding the particles together comprising a ROMP polymer or co-polymer having oligonucleotides bound thereto, the oligonucleotides bound to the ROMP polymer or co-polymer having a sequence complementary to at least a portion of the sequence of the oligonucleotides bound to the particles.
  • the oligonucleotides bound to the particles may have a spacer portion for attaching the oligonucleotides to the particles and a recognition portion that has a sequence that is complementary to at least a portion of the sequence of another oligonucleotide.
  • materials or structures are provided that are comprised of:
  • oligonucleotides comprising at least one type of recognition oligonucleotides, each type of recognition oligonucleotides comprising a spacer portion and a recognition portion, the spacer portion having a functional group through which the spacer portion is bound to the particles, the recognition portion having a sequence complementary to at least one portion of the sequence of another oligonucleotide;
  • a connector for holding the particles together comprising a ROMP polymer or co-polymer having oligonucleotides bound thereto, the oligonucleotides comprise a spacer portion and a recognition portion, wherein the spacer portion is bound to the ROMP polymer or co-polymer and the recognition portion has a sequence complementary to at least one portion of the sequence of the oligonucleotides bound to the particles.
  • materials or structures are provided that comprise:
  • each type of recognition oligonucleotides comprising a spacer portion and a recognition portion, the spacer portion having a functional group through which the spacer portion is bound to the particles, the recognition portion having a sequence complementary to at least one portion of the sequence of another oligonucleotide;
  • a connector for holding the particles together comprising a ROMP polymer or co-polymer having oligonucleotides bound thereto, the oligonucleotides comprise a spacer portion and a recognition portion, wherein the spacer portion is bound to the ROMP polymer or co-polymer and the recognition portion having a sequence complementary to at least one portion of the sequence of the oligonucleotides bound to the particles.
  • materials or structures are provided that comprise:
  • oligonucleotide polymer conjugates for holding the particles together, the oligonucleotide polymer conjugate comprising a ROMP polymer having oligonucleotides bound thereto, the oligonucleotides of the oligonucleotide polymer conjugate having a sequence complementary to at least one portion of the sequence of a second type of oligonucleotides bound to the first type of particles.
  • the particles in the materials or structures comprise metallic, particles, semiconductor particles, polymer latex particles, inorganic particles or a combination thereof.
  • the metallic particles may be made of gold, and the semiconductor particles may be made of CdSe/ZnS (core/shell).
  • the polymer latex particles may be composed of polyacrylates and the inorganic particles may be comprised of silica or metal oxide. Prefenably the particles are nanoparticles.
  • the spacer portion of the oligonucleotides bound to he ROMP polymer or co-polymer comprises from about 4 to about 30 nucleotides, preferably about 4 nucleotides.
  • the spacer portion of the oligonucleotides bound to the particles generally range between about 10 to about 30 nucleotides, preferably at least 10 nucleotides.
  • a method of fabrication comprises: providing a ROMP polymer or co-polymer having at least one type of oligonucleotides bound thereto, the oligonucleotides having a selected sequence, the sequence of each type of oligonucleotide having at least two portions; providing one or more types of particle-oligonucleotide conjugates, the oligonucleotides attached to the particles of each of the types of conjugates having a sequence complementary to the sequence of a portion of a oligonucleotide bound to the ROMP polymer or co-polymer; and contacting the ROMP polymer or co-polymer and particle oligonupcleotide conjugates under conditions effective to allow hybridization of the oligonucleotides attached to the particles to the oligonucleotides bound to the ROMP polymer or co-polymer so that a desired material or structure is formed wherein the
  • a method of fabrication comprises: providing at least two types of particle-oligonucleotide conjugates, the first type of particle-oligonucleotide conjugates have at least two types of oligonucleotides wherein the first type of oligonucleotides attached to the first type of particle-oligonucleotide conjugates has a sequence that is complementary to that of the oligonucleotides attached to the particles of the second type of conjugates, the second type of oligonucleotides attached to the particles of the first type of conjugates having a sequence that is complementary to that of the oligonucleotides attached to the particles of a second type of conjugates; providing a ROMP polymer or co-polymer having oligonucleotides bound thereto, the oligonucleotides having a sequence that is complementary to a second type of oligonucleotides bound to the first type of particle-oligonucleotide conjugates
  • a method of fabrication comprises: providing first and second ROMP polymers or co-polymers having oligonucleotides bound thereto, the oligonucleotides bound to the first ROMP polymer or co-polymer having a sequence that is complementary to the oligonucleotides bound to the second ROMP polymer or co-polymer; and contacting the first and second ROMP polymers or co-polymer under conditions effective to allow hybridization of the oligonucleotides on the first ROMP polymer or co-polymer with the oligonucleotides on the second ROMP polymer or co-polymer so that a desired material or structure is formed.
  • the present invention also provides a method for preparing a ROMP polymer or co-polymer having oligonucleotides bound thereto.
  • the method comprises: providing (i) a ROMP polymer or co-polymer modified with chlorophosphoramidite and (ii) oligonucleotides bound to a solid support; contacting the chlorophosphoramidite-modified ROMP polymer with the oligonucleotides bound to a support to produce an oligonucleotide ROMP polymer conjugate bound to the support; and cleaving the oligonucleotide-modified ROMP polymer or copolymer from the support.
  • the present invention also provides a method for the detection of one or more target nucleic acids in a sample, the sequence of each nucleic acid having at least two portions.
  • the method comprises: providing one or more types of oligonucleotide-modified ROMP polymer or copolymer, the sequence of the oligonucleotides bound to each type of polymer or copolymer has at least two portions wherein at least one portion of the sequence of the oligonucleotides is complementary to first portion of a sequence of a target nucleic acid, wherein the oligonucleotides bound to one type of polymer or copolymer is different from another type, wherein each type of polymer or copolymer serves as a unique identifier for a particular target nucleic acid, and wherein the polymer or copolymer includes electrochemical labels; providing a gold electrode surface having oligonucleotides bound thereto, the oligonucleotides that are bound to the surface
  • the ROMP polymer or copolymer are chemically defined and includes a defined number of electrochemical labels. Moreover, electrochemical detection may occur using cyclic voltammetry or differential pulse voltammetry.
  • the surface may have a plurality of types of oligonucleotides attached thereto in an array to allow for the detection of multiple different nucleic acid targets.
  • the sample may be first contacted with the surface so that one or more target nucleic acids hybridizes with complementary oligonucleotides bound to the surface and then the target nucleic acids bound to the surface is contacted with the polymer or copolymer so that at least some of the oligonucleotides bound to the polymer or copolymer hybridize with a portion of the sequence of the target nucleic acid bound to the surface.
  • the polymer or copolymer is contacted with the sample so that at least some of the oligonucleotides bound to the polymer or copolymer hybridize with a portion of the sequence of the target nucleic acids; and contacting the target nucleic acids bound to the polymer or copolymer with the surface so that a portion of the sequence of the target nucleic acids bound to the polymer or copolymer hybridizes with complementary oligonucleotides bound to the surface.
  • the sample, polymer or copolymer, and surface are contacted simultaneously.
  • signal amplification may be performed by providing a second oligonucleotide-modified ROMP polymer or copolymer, the sequence of the oligonucleotides bound to the second polymer or copolymer has at least two portions wherein at least one portion of the sequence of the oligonucleotides bound to the second polymer or copolymer is complementary to oligonucleotides bound to the first oligonucleotide-modified ROMP polymer or co-polymer; and contacting the second ROMP polymer or co-polymer with the one or more types of the first ROMP polymer or copolymer bound to the surface.
  • Further signal amplication may be achieved by further providing a third oligonucleotide- modified ROMP polymer or copolymer, the sequence of the oligonucleotides bound to the second polymer or copolymer has at least two portions wherein at least one portion of the sequence of the oligonucleotides bound to the second polymer or copolymer is complementary to oligonucleotides bound to the first oligonucleotide-modified ROMP polymer or co-polymer; and contacting the third ROMP polymer or co-polymer with the second ROMP polymer or copolymer bound to the surface.
  • kits for detecting one or more target nucleic acids in a sample comprising at least one or more containers including one or more types of chlorophosphoramidite modified ROMP polymer or copolymer, wherein each polymer or copolymer has a different redox activity and can be used for coupling with oligonucleotides.
  • the kit comprising at least one or more containers including one or more types of chlorophosphoramidite-modifiable ROMP polymer or copolymer, wherein each polymer or copolymer has a different redox activity and can serve as an identifier for a specific target nucleic acid.
  • the kit comprising at least one or more containers including one or more types of oligonucleotide-modified ROMP polymer or copolymer, wherein each polymer or copolymer has a different redox activity and serves as an identifier for a specific target nucleic acid.
  • the present invention also provides a system for detecting one or more target nucleic acids in a sample, the sequence of target nucleic acids have at least two portions, in a sample comprising
  • oligonucleotide-modified ROMP polymer or copolymer wherein each polymer or copolymer has a different redox activity and serves as an identifier for a specific target nucleic acid, the oligonucleotides bound to one type of polymer or copolymer is different from another, the oligonucleotides have a sequence having at least two portions, one portion of the sequence of the oligonucleotides is complementary to a first portion of a target nucleic acid;
  • a gold electrode surface having oligonucleotides bound thereto wherein the oligonucleotides bound to the surface has a sequence that is complementary to a second portion of a target nucleic acid;
  • Two complementary DNA- modified ROMP polymers were prepared: 3'-GCG TAA GTC CTA A 10 -5'- ⁇ oly2 (Hybrid I) and 3 '-TAG GAC TTA CGC A10-5'-poly2 (Hybrid II).
  • FIG. 3 A. UN-Vis spectrum of Hybrid-I.
  • Figure 4 Synthetic scheme illustrating preparation of a D ⁇ A-modified ROMP Dlock co-polymer from monomer 2 and monomer 4 via ROMP polymerization using catalyst 1, post-polymerization of the poly2-block-poly4 co-polymer with chlorophosphoramidite 3 and coupling the modified ROMP polymer to D ⁇ A using solid >hase synthesis.
  • Figure 5. (A) The cyclic voltammogram of Hybrid IV in 0.2 M [(n-Bu) N]PF 6 in CH 2 C1 2 .
  • Figure 6 Synthetic scheme illustrating preparation of a redox-active DNA- modified ROMP block polymers from monomer 2 and a norbornenyl-modified fenocene monomer in the presence of catalyst 1, modifying the resultant ROMP polymer using chlorophosphoramidite 3 and coupling the modified ROMP polymer to DNA using solid phase synthesis. Suitable, but non-limiting, examples of norbornenyl-modified fenocene monomers are illustrated therein.
  • FIG. 7 A. The UN- Vis absorption spectrum of D ⁇ A/ROMP polymer hybrids in water. B. UN-Nis absorption spectrum of purified Hybrid I. One major peak at 25 min was observed at both 260 and 310 nm, indicating that D ⁇ A is coupled to polymer backbone.
  • Figure 9 Scheme illustrating examples of redox active ROMP triblock copolymers having blocks that differ in size and type of norbornenyl-modified fenocene monomers.
  • FIG. 11 (A) UV-vis spectra of the solution containing complementary hybrid molecules (Hybrid Hybrid II) before and after DNA melting temperature. (B) Thermal denaturation curves of aggregates formed from hybrid molecules. A thermal denaturation curve for duplex DNA formed from oligonucleotides with same sequences as Hybrid I and II is given for comparison.
  • FIG. 12 DNA detection scheme using DNA-modified ROMP block copolymer probes.
  • Target nucleic acid sequence a'b' binds via portion a' to the complementary oligonucleotides a that are bound to the gold electrode surface.
  • the ROMP polymer having fenocenes as electrochemical tags and oligonucleotides b (complementary to b') bind to the nucleic acid.
  • Figure 13 Alternating Cunent (AC) voltammograms illustrating that gold electrodes treated with complementary target nucleic acid sequence produced a detectable signal while no signal was detected in the absence of complementary target.
  • Figure 14 Scheme illustrating the UV spectrum of oligonucleotide-modified ROMP polymer before and after Centricon-50 ultrafiltration.
  • Figure 15 Scheme illustrating signal amplification of an complex of oligonucleotide-modified ROMP co-polymer, a target nucleic acid, and oligonucleotides bound to a gold electrode surface as shown in Figure 12.
  • a second oligonucleotide b' (complementary to b)-modified ROMP co-polymer is hybridized to the complex to form a second complex.
  • a third oligonucleotide b (complementary to b')-modified ROMP copolymer is hybridized to the second complex.
  • ROMP has been used to generate defined, biologically active polymers (Gibson et al., Chem. Commun., 1095-1096 (1997); Biagini et al., Chem. Commun., 1097-1098 (1997); Biagini et al., Polymer, 39, 1007-1014 (1998); and Kiessling et al, Topics in Organometallic Chemistry, 1, 199-231 (1998)) with potent and unique activities that range from inhibiting protein-carbohydrate recognition events to promoting the proteolytic release of cell surface proteins (Mortell et al., J. Am. Chem. Soc, 118, 2297- 2298 (1996); Mortell et al., J. Am. Chem.
  • ROMP polymers have a number of advantages.
  • the ROMP reaction can be performed under living polymerization conditions, and if the rate of initiation is faster than that of propagation, varying the monomer to initiator ratio (M:I) can generate materials of defined length (Ivin and Mol, Olefin Metathesis and Metathesis Polymerization, 2 nd . Ed.; Academic Press: San Diego, 1997).
  • M:I monomer to initiator ratio
  • ruthenium metal carbene initiators are tolerant of a wide range of functional groups.
  • ROMP polymerization reaction conditions and any suitable metathesis catalyst may be used to prepare the ROMP polymer or co-polymers used a templates to prepare the oligonucleotide-modified ROMP polymers or copolymers.
  • the parameters for the ROMP polymerization reactions used in the present invention such as the atmosphere, choice of catalyst, the ratio of catalyst to monomer, the reaction temperatures, the solvents that may be used, the additives and other agents that may be present during the polymerization reaction, and the methods for canying out the metathesis polymerization will vary and can be selected by one of ordinary skill in the art without undue experimentation. Many suitable conditions and parameters are described, for instance, in Schwab et al., J. Am. Chem.
  • the polymerization of the olefin is carried out by adding the metathesis catalyst to a solution of the monomer starting material which has been heated to an initial reaction temperature.
  • the catalyst may be first added to the monomer starting material and the mnixture then heated to the required temperature.
  • the initial reaction temperature is not critical; but, as is known, this temperature does affect the rate of the polymerization reaction.
  • the reaction temperature will be in the range of about 0° C. to about 100° C, and preferably about 25° C to about 45° C.
  • the reaction is generally carried out under an inert atmosphere (e.g., nitrogen or argon). Pressure is not critical, but may be varied to maintain a liquid phase reaction mixture. Reaction times can vary from several minutes to several days.
  • the ratio of catalyst to starting material is not critical and can within the range from about 1:5 to about 1:200,000 by mole. Ratios of catalyst to starting material of between about 1:2,000 and 1:15,000 by mole are prefened.
  • the invention may be practiced using catalyst/starting material ratios outside of the above ranges.
  • the monomer starting material may optionally be refluxed, either in a solution or by itself, run through absorption purification, and degassed before the catalyst is added; although, none of these procedures is necessary in practicing the invention.
  • solvents that maybe used include organic, protic, or aqueous solvents which are inert under the reaction conditions.
  • suitable solvents may include aromatic hydrocarbons, chlorinated hydrocarbons, ethers, alipabtic hydrocarbons, alcohols, water, etc which are unreactive under the reaction conditions. Specific examples include 1,2-dichloroethane, benzene, toluene, p-xylene, methylene chloride, dichlorobenzene, tetrahydrofuran, diethylether, pentane, methanol.
  • the polymer is generally terminated by reacting the catalyst with a capping agent.
  • the capping agent is typically matched to the catalyst.
  • ruthenium catalyst for example, ethyl vinyl ether has been used.
  • ROMP can provide polymers of varying average lengths (i.e. varying degree of polymerization, DP) depending on the ratio of monomer to ROMP catalyst (i.e., initiator).
  • Suitable catalysts include, but are not limited to, Grubb's ruthenium metal carbene catalyst (Compound 41, Fig. 6) and the compounds shown in Fig. 3 and disclosed in Kingsbury et al., J. Amer. Chem. Soc, 121, 791-799 (1999); Schwab et al., J. Amer. Chem. Soc, 118, 100-110 (1996); Dias et al., Organometallics, 17, 2758-2767 (1998); del Rio et al., Tetrahedron Lett., 40, 1401-1404 (1999); Furstner et al., Chem. Commun., 95-96 (1999); Weskamp et al., Angew.
  • suitable catalysts are ruthenium and osilum carbene complex catalysts disclosed in the above cited references.
  • the prefened ruthenium and osmium carbene complex catalysts include those which are stable in the presence of a variety of functional groups including hycdroxyl, thiol, thioetlher, ketone, aldehyde, ester, ether, amine, imine, amide, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, peroxy, anhydride, carbamate, and halogen.
  • the starting monomers, impurities in the monomer, any substituent groups on the catalyst, and other additives may include one or more of the above listed groups without deactivating the catalysts.
  • the catalyst preferably includes a ruthenium or osmium metal center that is in a +2 oxidation state, has an electron count of 16, and is pentacoordinated.
  • ruthenium or osmium carbene complex catalysts may be represented by the formula:
  • M is O or Ru
  • R and R 1 may be the same or different and may be hydrogen or a substituent group which may be C 2 -C20 alkenyl, C2 -C20 alkynyl, Ci-C2o alkyl, aryl, Ci -C20 carboxylate, -C20 alkoxy, C2 -C 2 o alkenyloxy, C 2 -C 20 alkynyloxy, aryloxy, C2-C20 alkoxycarbonyl, Ci -C20 alkylthio, Ci -C2 0 alkylsulfonyl and Ci -C 20 alkylsulfiniyl.
  • the substituent group may be substituted with one or more groups selected from Ci -C 5 alkyl, halide, Ci - C 5 alkoxy, and phuenyl.
  • the phenyl group may optionally be substituted wixTh one or more groups selected from halide, Ci-C 5 alkyl, and Ci -C 5 alkoxy.
  • the substituent group may be substituted with one or more functional groups selected from hydroxyl, thiol, thioethler, ketone, aldehyde, ester, ether, amine, imine, amide, nitro, carboxylic acid, disulfide carbonate, isocyanate, carbodiimide, carboal koxy, peroxy, anhydride, carbamate, and halogen.
  • one or more functional groups selected from hydroxyl, thiol, thioethler, ketone, aldehyde, ester, ether, amine, imine, amide, nitro, carboxylic acid, disulfide carbonate, isocyanate, carbodiimide, carboal koxy, peroxy, anhydride, carbamate, and halogen.
  • R and R 1 are the same or different and may be hydrogen, substituted aryl, unsubstituted aryl, substituted vinyl, and unsubstituted vinyl; where the substituted aryl and substituted vinyl are each substituted with one or more groups selected from hydroxyl, thiol, thioether, ketone, aldehyde, ester, ether, amine, imine, amide, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, peroxy, anhydride, carbamate, and halogen, Ci -C 5 allcyl, C ⁇ -C 5 alkoxy, unsubstituted phenyl, and phenyl substituted with halide, Ci -C 5 alkyl or Ci -C 5 alkoxy;
  • X and X 1 may be the same or different and may generally be hydrogen or any anionic liganid.
  • An anionic ligand is any ligand which when removed from a metal center in its closed shell electron configuration has a negative charge.
  • X and X 1 are thre same or different and may be halogen, hydrogen or a substituent group selected from C ⁇ -C20 alkyl, aryl, Ci -C20 alkoxide, aryloxide, Ci -C 2 o alkyldiketone, aryldiketonate, Ci -C 2 0 carboxylate, aryl or Ci -C20 allcylsulfonate, Ci-C 2 o alkylthio, Ci -C 2 o alkylsulfonyl, and Ci -C 20 alkylsulfinyl.
  • the substituent groups may optionally be substituted with Ci -C5 alkyl, halogen, Ci -C5 akloxy or phenyl.
  • the phenyl may be optionally substituted with halogen, Ci -C 5 alkyl, or d -C 5 alkoxy.
  • X and X 1 are the same or different and may be CI, Br, I, H or a substituent group selected from benzoate, Ci -C 5 carboxylate, Ci -C 5 alkyl, phenoxy, Ci -C 5 alkoxy, Ci -C 5 alkylthio, aryl, and Ci -C 5 alkyl sulfonate.
  • the substituent groups may be optionally substituted with Ci -C 5 alkyl or a phenyl group.
  • the phenyl group may optionally be substituted with halogen, Ci -C 5 alkyl or Ci -C 5 alkcoxy.
  • X and X 1 are the same or different and are selected from CI, CF 3 CO 2 , CH 3 CO 2 , CFH 2 CO 2 , (CH 3 ) 3 CO, (CF 3 ) 2 (CH 3 )CO, (CF 3 ) (CH 3 ) 2 CO, PhO, MeO, EtO, tosylate, mesylate, and trifluoromethanesulfonate.
  • X and X 1 are both CI; and L and L 1 may be the same or different and may be generally be any neutral electron donor.
  • a neutral electron donor is any ligand which, when removed from a metal center in its closed shell electron configuration, has a neutral charge.
  • L and L 1 may be the same or different and may be phosphines, sulfonated phospines, phosphites, phiosphinites, phosphonites, arsines, stibines, ethers, amines, amides, sulfoxides, carboxyls, nitrosyls, pyridines, and thioethers.
  • L and L 1 are the same or different and are phosphines of the formula PR 3 R 4 R 5 where R 3 is a secondary alkyl or cycloaklyl and R 4 and R 5 are the same or different and are aryl, Ci -Cio primary alkyl, secondary alkyl, or cycloalkyl.
  • L and L are the same or different and are ⁇ P(cyclohexyl) 3 , ⁇ P(cyclopentyl) 3 , or ⁇ P(isopropyl) 3 .
  • L and L 1 may also be ⁇ P(phenyl) 3 .
  • a prefened group of catalysts are those where M is Ru; R 1 and R are independently hydrogen or substituted or unsubstituted aryl or substituted or unsubstituted vinyl; X and X 1 are CI; and L and L 1 are triphenylphosphines or trialkylphosphines such as tricyclopentylphosphine, tricyclohexylphosphine, and triisopropylphosphine.
  • the substituted aryl and substituted vinyl may each be substituted with one or more groups including C ⁇ -C 5 alkyl, halide, d -C 5 alkoxy, and a phenyl group which may be optionally substituted with one or more halide, Ci -C 5 alkyl, or Ci - C5 alkoxy groups.
  • the substituted aryl and substituted vinyl may also be substituted with one or more functional groups including hydroxyl, thiol, thioether, ketone, aldehyde, ester, ether, amine, imine, amide, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, peroxy, anhydride, carbamate, and halogen.
  • one or more functional groups including hydroxyl, thiol, thioether, ketone, aldehyde, ester, ether, amine, imine, amide, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, peroxy, anhydride, carbamate, and halogen.
  • Particularly prefened catalysts can be represented by the formulas:
  • Cy is cyclopentyl or cyclohexyl
  • Ph is phenyl
  • the most prefened catalysts can be represented by the formula:
  • Cy is cyclopenotyl or cyclohexyl
  • Ph is phenyl
  • the catalysts described above are useful in polymerization of a wide variety of olefin monomers through metathesis polymerization, particularly ROMP of cycloolefins.
  • Suitable monomers include olefins that can be polymerized by any of the ruthenium or osmium metathesis polymerization catalysts that were discussed arbove.
  • Suitable monomers for use in the present invention have at least one polymerizable group (and often only one polymerizable group) and at least one functional group (used for subsequent modification for coupling to an oligonucleotide) and/or reporter label and result in a polymer or polymer template that is stable to the ROMP polymerization conditions.
  • the olefin monomers may be unfunctionalized or functionalized to contain one or more functional groups selected from the group consisting of hydroxyl, thiol, thioether, ketone, aldehyde, ester, ether, amine, imine, amidie, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, peroxy, anhydride, carbamate, and halogen.
  • the olefin may be a strained cyclic olefin, or unstrained cyclic olefin, each of which may be functionalized or unfunctionalized.
  • Prefened monomers include functionalized or unfunctionalized cyclic olefins that are polymerized through ROMP reactions. This polymerization process includes contacting a functionalized or unfunctionalized cyclic olefin with a ruthenium or osmium metathesis catalysts discussed above.
  • the cyclic olefins may be strained or unstrained and may be monocyclic, bicyclic, or multicyclic olefins.
  • the cyclic olefin may contain one or more functional groups including hydroxyl, thiol, thioether, ketone, aldehyde, ester, ether, amine, imine, amide, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, peroxy, anhydride, carbamate, and halogen.
  • Suitable cyclic olefin monomers include monomers disclosed in U.S. Pat. No. 1,943,621 to Janda, et al., U.S. Pat. No. 4,324,717 to Layer, and U.S. Pat. No. 4,301,306 o Layer, all of which are herein inco ⁇ orated by reference.
  • Suitable cyclic olefin monomers include norbornene-type monomers which are characterized by the presence of at least one norbornene group which can be substituted >r unsubstituted.
  • Suitable norbornene type monomers include substituted norbornenes md unsubstituted norbornene, dicyclopentadiene, di(methyl) dicyclopentadiene, lilhydrodicyclopentadiene, cyclopentadiene trimers, tetramers of cyclopentadiene, etracyclododecene, and substituted tetracyclododecenes.
  • Common norbornene-type monomers can be represented by the following formulas:
  • R and R 1 may be the same or different and may be hydrogen or a substitute group which may be a halogen, Ci -Ci 2 alkyl groups, C 2 -Cu alkylene groups, C 6 -Cn cycloalkyl groups, C 6 -C 12 cycloalkylene groups, and Ci -Ci 2 aryl groups or R and R 1 together form saturated or unsaturated cyclic groups of from 4 to 12 carbon atoms with the two ring carbon atoms connected thereto, said ring carbon atoms forming part of and contributing to the 4 to 12 carbon atoms in the cyclic group.
  • R and R 1 have the same meaning as indicated above and n is greater than 1.
  • monomers suitable for use in this invention include: ethylidenenorbornene, methyltetracyclododecene, methylnorborinene, ethylnorbornene, dimethylnorbornene and similar derivatives, norbornadiene, cyclopentene, cycloheptene, cyclooctene, 7-oxanorbornene, 7-oxanorborniene derivatives, 7-oxabicyclo[2.2.1]hept- 5ene derivatives, 7-oxanorbornadiene, cyclododecene, 2-norbornene, also named bicyclo[2.2.1]-2-heptene and substituted bicyclic norbornenes, 5-methyl-2-norbornene, 5,6-dimethyl-2-norbornene, 5-ethyl-2-norbornene, 5-butyl-2-norbornene, 5-hexyl-2- norbornene,
  • the cyclic olefin is cyclobutene, dimethyl dicyclopentadiene, cyclopentene, cycloheptene, cyclooctene, cyclononene, cyclodecene, cyclooctadiene, cyclononadiene, cyclododecene, norbornene, norbornadiene, 7- oxanorbornene, 7-oxanorbornadiene, and dicyclopentadiene; each of which may be functionalized or urifunctionalized.
  • the cyclic olefin is subsitututed norbomenes.
  • the norbomenes include a functional group and/or reporter label that is attached to the norbornene via a linker.
  • Any suitable linker of any suitable length may be used, including without limitation, linear or branched C1-C20 alkyls, Ci-C2o alkyl ethers, aryl Ci-C2o alkyls, aryl Ci-C 2 o alkyls ethers, C1-C20 alkenyls, Ci-C2o alkynyls, aryl C1-C20 alkynyls which may optionally substituted.
  • a suitable reporter label may be attached including without limitation UV labels, fluorescent labels, radiolabels, redox labels, etc.
  • a prefened monomer is a norbornenyl-substituted fenocene.
  • the monomer is a norbornene bound to a fenocene via a linker.
  • a prefened norbornenyl- substituted fenocene (NSF) monomer has the following formula:
  • linker R may be any suitable moiety for connecting the fenocene or any other electrochemical tag to the norbornene structure.
  • 2 and R 3 may be independently H, halogen, -OH, or C 1 -C 20 alkyl or alkoxide.
  • the invention contemplates the preparation of ROMP homopolymers, as well as random and block co-polymers, te ⁇ olymers, random copolymers with more than three different monomers, and multiblock copolymers of the suitable monomers discussed above.
  • oligonucleotide-modified ROMP block co-polymers may be designed with highly tailorable redox-activities. Based on the block co-polymer strategy described in the Examples below, one can inco ⁇ orate about four different NSF monomers as indicators.
  • multiblock e.g., triblock, co-polymers containing different NSF derivatives
  • a ratio between the redox active blocks during the ROMP reaction one can generate many many different indicators rather than four in the case of diblock co-polymers which is useful for multichannel DNA detection.
  • polymers prepared with monomers having functional groups for subsequent modification these polymers may be used as templates for a post-polymerization reaction with a chlorophosphoramidite reagent under suitable conditions to produce a modified template suitable for coupling to DNA using standard DNA solid phase synthetic techniques.
  • a chlorophosphoramidite reagent Any chlorophosphoramidite reagent and any suitable modification conditions may be used to prepare the chlorophosphoramidite modified polymer template.
  • chlrophosphoramidite reagents include 2-cyanoethyl diisopropylchlorophosphoramidite or 2-cyanoethyl tetraisopropylchlorophosphoramidite.
  • oligonucleotides are readily attached to the polymer backbone to produced novel oligonucleotide-modified ROMP polymers or co-polymers have a well- defined polymer structure.
  • three dimensional aggregated structures comprised of ROMP polymers or co-polymers having complementary oligonucleotides can be produced and have extended hybridization networks which precipate reversibly from aqueous solution. This establishes that the attachment of the oligonucleotides to the polymers and existence of one or more blocks in the co-polymers do not interfere with the recognition properties of the DNA.
  • oligonucleotide-modified polymers or copolymers for detection of target nucleic acids or other oligonucleotides and the further preparation of new materials.
  • monomers such as norbornene linked to electrochemically active molecules can be used for preparing oligonucleotide-modified ROMP block copolymers with electrochemical tags and having redox activity that can be used for the electrochemical detection of target nucleic acids or other oligonucleotides using cyclic voltammetry or pulse voltammetry. See for instance Figure 12.
  • a complementary oligonucleotide-modified ROMP block copolymer may be used to bind to any unbound oligonucleotide bound to the ROMP block copolymer involved in the initial complexation with a target nucleic acid and oligonucleotides bound to the gold electrode surface. See Figure 14.
  • DNA hybridization interactions between oligonucleotide-modified ROMP polymers or copolymers with complementary oligonucleotides labeled particles may be exploited to prepare materials with new properties.
  • particles including, without limitation, latex particles, polystrene particles, and particles such as metallic particles (e.g., gold), semiconductor particles (e.g., CdSe/ZnS core/shell), insulator particles, polymer particles (e.g., polyacrylates), inorganic particles (e.g., silica or metal oxide) or combinations there of.
  • suitable particles and methods for preparation are described, for instance, in Mirkin U.S. Patent No.
  • the present invention contemplates the use of any suitable particle having oligonucleotides attached thereto that are suitable for use in detection assays. In practicing this invention, however, nanoparticles are prefened. The size, shape and chemical composition of the particles will contribute to the properties of the resulting probe including the DNA barcode. These properties include optical properties, optoelectronic properties, electrochemical properties, electronic properties, stability in various solutions, pore and channel size variation, ability to separate bioactive molecules while acting as a filter, etc.
  • suitable particles include, without limitation, nano- and microsized core particles, aggregated particles, isotropic (such as spherical particles) and anisotropic particles (such as non-spherical rods, tetrahedral, prisms) and core-shell particles such as the ones described in U.S. Patent application no. 10/034,451, filed December 28, 2002 and International application no. PCT/USOl/50825, filed December 28, 2002, which are inco ⁇ orated by reference in their entirety.
  • Particles useful in the practice of the invention include metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., fenomagnetite) colloidal materials.
  • Other particles useful in the practice of the invention include ZnS, ZnO, Ti ⁇ 2, Agl, AgBr, Hgl 2 , PbS, PbSe, ZnTe, CdTe, hi2S 3 , In 2 Se 3 , Cd P 2 , Cd 3 As 2 , hiAs, and GaAs.
  • the size of the particles is preferably from about 5 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm.
  • the particles may also be rods, prisms, or tetrahedra.
  • Suitable particles are also commercially available from, e.g., Ted Pella, Inc. (gold), Amersham Co ⁇ oration (gold) and Nanoprobes, Inc. (gold).
  • Gold colloidal particles have high extinction coefficients for the bands that give rise to their beautiful colors. These intense colors change with particle size, concentration, inte ⁇ article distance, and extent of aggregation and shape (geometry) of the aggregates, making these materials particularly attractive for colorimetric assays. For instance, hybridization of oligonucleotides attached to gold particles with oligonucleotides and nucleic acids results in an immediate color change visible to the naked eye (see, e.g., the Examples).
  • the D ⁇ A/polymer conjugate was utilized to form particle assemblies.
  • a PBS buffer solution of Hybrid-I (12 ⁇ L of 8.3 ⁇ M in D ⁇ A) is mixed with a PBS buffer solution of 13 nm Au particles (260 ⁇ L of 9.7 nM in particle) modified with complementary D ⁇ A strands
  • 2 the formation of three-dimentional particle aggregates is signaled by the diagnostic shift in the surface plasmon resonance of the particles (from 520 nm to 570 nm, Figure 3C) and a conesponding change in color (from red to pu ⁇ le).
  • 2 ' 3 Transmission electron microscopy studies reveal a high networked aggregate (Figure 3D). Control experiments in which a solution of the same particles was mixed with a buffered solution of Hybrid-II (which is non-complementary) resulted in no aggregate formation under nearly identical conditions.
  • a or “an” entity refers to one or more of that entity.
  • a characteristic refers to one or more characteristics or at least one characteristic.
  • the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” have been used interchangeably.
  • GPC Gel permeation chromatography
  • GPC was also carried out on a Waters Breeze system equipped with a 1525 HPLC pump, a 2487 Dual Wavelength Absorbance Detector, a 2410 Refractive Index Dectector, and a Shodex GPC Mixed-Bed KF-806-L column connected in series with a Shodix GPC Mixed-Bed KF-803-L (column dimensions 300 mm x 8 mm for both).
  • UV- vis spectra were recorded using a Hewlett Packard (HP) 8452A diode-anay spectrophotometer. Transmission electron microscopy was performed on a Hitachi 8100 microscope. A typical sample was prepared by dropping 10 uL of aggregate solution onto a holey carbon TEM grid, followed by wicking the solution away. The grid was subsequently dried and imaged. Electronic abso ⁇ tion spectra were recorded using a Hewlett Packard (HP) 8452A diode anay spectrophotometer.
  • the supporting electrolyte was 0.2 M [(n-Bu) 4 N]PF 6 in CH 2 C1 2 , and all experiments were carried out at room temperature after the solution was degassed by purging with nitrogen for 10 min.
  • Alternating Cunent (AC) voltammograms were acquired in a low frequency (10 Hz) mode at a peak AC voltage implitude of 25 mV.
  • catalyst Cl 2 Ru(PCy 3 ) 2 CHPh 18 (l)and 5-e o-norbornen-2-ol ⁇ , 2 0 vere prepared from literature procedures.
  • catalyst 1 can also be purchased rom Strem Chemicals.
  • the synthesis and purification of (alkanethiol)-modified tligonucleotides was performed as described elsewhere.
  • Acetonitrile, CHC1 3 , NEt 3 , and ⁇ 2 CI 2 were distilled over calcium hydride.
  • Tefrahydrofuran (THF) toluene, and diethyl ther were distilled over sodiiim/benzophenone.
  • Methanol was distilled over Mg(OMe) 2 .
  • This Example describes the preparation of a ROMP homopolymer from a norbornene monomer 2 possessing a UV tag followed by post-polymerization modification of the polymer to attach oligonucleotides using standard DNA solid phase synthetic techniques.
  • exo-5-norbornene-2-(4-iodobenzyloxy) (B).
  • exo-5-norbornene-2-ol (1.00 g, 9.08 mmol) was weighed into a 50- mL Schlenk flask.
  • THF 15 mL was added, and the solution was stined while oil-free sodium metal (300 mg, 13.0 mmol) was added.
  • the mixture was then taken out of the glovebox, refluxed for 12 h under a nitrogen bubbler, and allowed to cool to room temperature.
  • reaction mixture Upon cooling to room temperature, the reaction mixture was poured into ether (50 mL) and washed successively with water (50 mL), 0.1 M NaOH (50 mL), 1.0 M HCl (50 mL), and brine (50 mL). The organic layer was collected, dried over sodium sulfate, and filtered into a 500-mL round bottom flask. The solvent was removed under vacuum. Column chromatography on silica gel with 30% CH 2 CI 2 in hexanes as the eluent gave the desired product B (1.75 g, 5.36 mmol, 94 %) as a clear oil.
  • PBS 0.3 M ⁇ aCl, 10 mM phosphate, pH 7
  • hybridization triggers the formation of an extended network aggregate of linked polymers, which is signaled by the immediate formation of a white precipitate. Presumably, this occurs because each polymer is modified with more than one strand of D ⁇ A, which leads to cooperative binding between many D ⁇ A-functionalized complementary polymer stands, as evidenced by the sha ⁇ melting transition (inset of Figure 3B). As expected, this hybridzation process is thermally reversible.
  • Example 2 the oligonucleotide modified ROMP polymer conjugate described in Example 1 was utilized to form nanoparticle assemblies.
  • a PBS buffer solution of Hybrid-I (12 ⁇ L of 8.3 ⁇ M in D ⁇ A) is mixed with a PBS buffer solution of 13 nm gold nanoparticles (260 ⁇ L of 9.7 nM in particle) modified with complementary DNA strands
  • 2 three-dimentional particle aggregates is signaled by the diagnostic shift in the surface plasmon resonance of the particles (from 520 nm to 570 nm, Figure 3C) and a conesponding change in color (from red to pu ⁇ le).
  • 2 ' 3 Transmission electron microscopy studies reveal a high networked aggregate (Figure 3D). Control experiments in which a solution of the same nanoparticles was mixed with a buffered solution of Hybrid-II (which is non-complementary) resulted in no aggregate formation under nearly identical conditions.
  • Oligonucleotide-modified 13 nm Au particles were prepared by literature methods (-110 oligonucleotides/particle). " Gold colloids (13 nm diameter) were prepared by reduction of HAuCl 4 with citrate as described by Frens, Nature Phys. Sci, 241, 20 (1973) and Grabar, Anal. Chem., 67, 735 (1995). Briefly, all glassware was cleaned in aqua regia (3 parts HCl, 1 part HNO 3 ), rinsed with nanopure H 2 O, then oven dried prior to use. HAuCLj and sodium citrate were purchased from Aldrich Chemical Company.
  • aqueous solution of HAuCl 4 (1 mM, 500 mL) was brought to a reflux while stirring, and then aqueous trisodium citrate (50 mL of a 38.8 mM solution) was added quickly, which resulted in a change in solution color from pale yellow to deep red. After the color change, the solution was refluxed for an additional fifteen minutes, allowed to cool to room temperature, and subsequently filtered through a 0.45 micron nylon filter (Micron Separations Inc.).
  • the resulting Au colloids solution were characterized by UN- Vis spectroscopy using a Hewlett Packard 8452A diode-anay spectrophotometer and by Transmission Electron Microscopy (TEM) using a Hitachi 8100 transmission electron microscope.
  • a typical solution of 13-nm diameter gold particles exhibited a characteristic surface plasmon band centered at 518-520 nm. Gold particles with diameters of 13 nm will produce a visible color change when aggregated with target and probe oligonucleotide sequences in the 10-72 nucleotide base pairs range.
  • 3'-thiol oligonucleotides Preparation of 3'-thiol oligonucleotides.
  • Thiol- Modifier C3 S-S CPG support was purchased from Glen Research and used in the automated synthesizer. The final dimethoxytrityl (DMT) protecting group was not removed to aid in purification.
  • DMT dimethoxytrityl
  • the supported oligonucleotide was placed in concentrated arnmoriium hydroxide (1 mL) for 16 hours at 55 °C to cleave the oligonucleotide from the solid support and remove the protecting groups from the bases.
  • the oligonucleotides were purified by preparative reverse-phase HPLC using an HP ODS Hypersil column (5 ⁇ m, 250 x 4 mm) with 0.03 M triethyl ammonium acetate (TEAA) eluent (pH 7) and a 1%/minute gradient of [95% CH 3 CN/5% 0.03 M TEAA] at a flow rate of 1 mL/minute, while monitoring the
  • the retention time of the DMT-protected modified 12- base oligomer averages ⁇ b/c it varies with the sequences> at about 30 minutes.
  • DMT was subsequently cleaved by soaking the purified oligonucleotide in an 80 % acetic icid solution for 30 minutes followed by evaporation.
  • the resulting oligonucleotide was
  • Dionex Nucleopac PA-100 column 250 x 4 mm
  • 10 mM NaOH (pH 12) eluent and a 2%/minute gradient of [10 mM NaOH, 1 M NaCl] at a flow rate of 1 mL/minute while monitoring the UV signal of DNA at 254 nm.
  • 5'-alkylthiol modified oligonucleotides were prepared using the following syringe method protocol: 1) a CPG- bound, detritylated oligonucleotide was synthesized on an automated DNA synthesizer (Expedite) using standard procedures; 2) the CPG-cartridge was removed and disposable syringes were attached to the ends; 3) 200 ⁇ L of a solution containing 20 ⁇ mole of 5- Thiol-Modifier C6-phosphoramidite (Glen Research) in dry acetonitrile was mixed with 200 ⁇ L of standard "tetrazole activator solution" and, via one of the syringes, introduced into the cartridge containing the oligonucleotide-CPG; 4) the solution was slowly pumped back and forth through the cartridge for 10 minutes and then ejected followed by washing with dry acetonitrile (2 x
  • the tritylated oligonucleotide derivative was then isolated and purified as described above for the 3'- alkylthiol oligonucleotides.
  • the trityl protecting group was then cleaved by adding 15 ⁇ L (for 10 OD's) of a 50 mM AgNO 3 solution to the dry oligonucleotide sample for 20 minutes, which resulted in a milky white suspension.
  • the excess silver nitrate was removed by adding 20 uL of a 10 mg/mL solution of DTT which immediately formed a yellow precipitate (within five minutes of reaction time) that was removed by centrifugation.
  • the solution was next centrifuged at 14,000 m in an Eppendorf Centrifuge 5414 for about 25 minutes to give a very pale pink supernatant containing most of the oligonucleotide (as indicated by the absorbance at 260 nm) along with 7-10% of the colloidal gold (as indicated by the absorbance at 520 nm), and a compact, dark, gelatinous residue at the bottom of the tube.
  • the supernatant was removed, and the residue was resuspended in about 200 ⁇ L of buffer [10 mM phosphate, 0.1 M NaCl] and recentrifixged.
  • This Example describes the preparation of a ROMP block co-polymer (poly2- block-poly4) modified with oligonucleotides. See Figure 2 for the chemical structures of the monomers and intermediates.
  • the polymer (poly2-block-poly4, 140 mg, 96%) was isolated by pouring the mixture into pentane, filtering, and repeatedly washing with fresh pentane (4 x 20 mL).
  • the data in this example illustrates that the post-polymerization modification of ROMP polymers and block copolymers with DNA can lead to DNA/polymer hybrid materials with a number of interesting properties associated with the hybrid structure.
  • the initial experiments described herein reveal that the recognition properties of the DNA strands are not adversely affected by attachment to the polymer. These new structures can be prepared with properties and function that depend upon the choice of ROMP monomer and DNA branch sites. Since the synthesis of block copolymers of 2 with other norbornenyl-modified compounds is a facile process, the isolation of other novel and potentially useful macromolecular hybrid materials should be readily accomplished by utilizing variations of the strategy presented herein.
  • Block Copolymers (Hybrid I-Hybrid IV) of DNA and redox active molecules (4 or 5) were synthesized from ROMP ( Figure 6). DNA sequences are given in Table 1.
  • the redox potential of the DNA/polymer hybrids can be tailored by using fenocene derivatives with electron donating or withdrawing substituent. Two different norbornenyl-modified fenocene derivatives, 4 and 5, were chosen because of their redox potential difference. Since the carbonyl group attached to the fenocene ring of 5, it oxidizes at higher potential than 4 ( ⁇ E1 /2 - 300 mV). Both monomers were polymerizable using 1 and inco ⁇ orated into the blockcopolymer structure to yield poly2- poly4 and poly2-poly5. Those polymer precursors were readily coupled to 5' end of oligonucleotides to yield Hybrid I-Hybrid TV as described in Figure 6.
  • 11-bromoundecanoyl-fenocene (2.00 g, 4.62 mmol) was dissolved in dry THF (15 mL) under nitrogen.
  • the cooled solution of deprotonated exo-5-norbornen- 2-ol was then transfened via cannula to the 11-bromoundecanoyl-fenocene solution with vigorous stirring.
  • the flask was capped with a water-cooled condenser and the resulting mixture was refluxed for an additional 12 h under a nitrogen bubbler.
  • reaction mixture Upon cooling to room temperature, the reaction mixture was poured into ether (50 mL) and washed successively with water (50 mL), 0.1 M NaOH (50 mL), 1.0 M HCl (50 mL), and brine (50 mL). The organic layer was collected, dried over sodium sulfate, and filtered into a 500-mL round bottom flask. The solvent from the filtrate was removed on a rotary evaporator. Column chromatography of the residue on silica gel with 10 % ethyl acetate in hexanes as the eluent gave the desired product 5 as a dark red oil.
  • reaction mixture was then poured into water and extracted with methylene chloride (2 x 50 mL). The organic portions were combined and washed with water (in 50 mL portions) until the aqueous washes become pH-neutral, dried over sodium sulfate, and filtered into a 500-mL round bottom flask. The solvent was removed from the filtrate and evaporated from the crude residue on a rotary evaporator. Column chromatography of the reaction residue on silica gel with 20% ethyl acetate in hexanes as the eluent gave the desired product as mixture of two isomers (1.502 g, 3.14 mmol, 57.6%) as a red oil.
  • reaction flask was then taken out of the glovebox, attached to a water-cooled condenser, refluxed for 12 h under a nitrogen bubbler, and allowed to cool to room temperature, h a separate 100- mL Schlenk flask, (3-bromopropyl)dibromofenocene (876 mg, 1.88 mmol) was dissolved in dry T ⁇ F (15 mL) under nitrogen.
  • the cooled solution of deprotonated exo- 5-norbornen-2-ol was then transfened via cannula to the (3- bromopropyl)dibromofenocene solution with vigorous stirring.
  • reaction flask was then taken out of the glovebox, attached to a water- cooled condenser, refluxed for 12 h under a nitrogen bubbler, and allowed to cool to room temperature.
  • tris(ethylene glycol)- ⁇ , ⁇ bis(p- tosylate) (2.10 g, 4.58 mmol) was dissolved in dry dioxane (15 mL) under nitrogen. This solution was then stined vigorously while the cooled solution of deprotonated exo-5- norbornen-2-ol was added via cannula.
  • the flask was capped with a water-cooled condenser and refluxed for an additional 48 h under a nitrogen bubbler.
  • the attachment of DNA to modified poly2-poly5 was accomplished using the syringe synthesis technique.
  • CDCI 3 was used as a solvent during the coupling of modified poly2-poly5 to the oligonucleotides on CPG support instead of acetonitrile.
  • the supported polymer was placed in 1 mL of ammonium hydroxide at 60 °C for 16 h to remove the protecting groups from bases and cleave the polymers and failure DNA stands from the support. Purification was accomplished using ultrafiltration with a Centricon-100 instrument.
  • HPLC High-performance liquid chromatography
  • Example 5 Synthesis of DNA-modified ROMP Triblock co-polymers. Based on the blockcopolymer strategy discussed above, this Example illustrates the preparation of DNA-modified ROMP triblock co-polymer one can inco ⁇ orate about four different indicators. To increase the number of indicators thus to use their redox potentials as a type of barcode, triblock copolymers were synthesized to contain two different fenocenyl derivatives, 4 and 6. By adjusting the ratio between any two redox active blocks, one can generate many different indicators rather than the maximum two in the case of diblock copolymers where each redox-active monomer can only be used once (Scheme 2).
  • Triblock copolymer precursors poly2-poly4-poly6 were synthesized by successively growing 2, 4 and 6 onto a propagating ROMP chain.
  • triblock copolymers with approximately 1:2 and 2:1 ratios of 4 and 6 were synthesized.
  • Gel permeation chromatography (GPC) data of these polymers showed a single peaks, indicating that the three components are in one entity.
  • GPC Gel permeation chromatography
  • Cyclic voltamograms of the polymers exhibit two peaks at 30 mV and 330 mV (versus Fc/Fc*) ( Figure 10A). Ratios of peak areas are 1:4.2 and X/X, respectively.
  • the mixture was stined for 12 h, then a solution of 6 (20 mg, 0.040 mmol) in dry THF (0.5 mL) was injected into the mixture.
  • the reaction was stined for 2 h, removed from the dry box, and the polymerization was terminated with ethyl vinyl ether (1 mL).
  • the polymer (poly2-poly4-poly6, 72 mg, 78%) was isolated by adding the mixture dropwise into a stined solution of pentane, filtering, and repeatedly washing with fresh pentane (4 x 20 mL).
  • HPLC High-performance liquid chromatography
  • a HP series 1100 HPLC equipped with an ion-exchange column with 10 mM NaOH eluent and a 2%/min gradient of [10 mM NaOH, 2 M NaCl] at a flow rate of 1 mL/min, while monitoring the UV absorbance at 260 nm and 310 nm.
  • Example 6 Preparation of Redox active DNA-modified ROMP random co-polymer
  • ROMP co-polymers were prepared in a random fashion, which might improve the solubility of the hybrid molecules, as a hydrophobic block in a hybrid molecule gets longer.
  • Random copolymers were synthesized from 5 and 7 by injecting 2, 5 and 7 at the same time rather than introducing them successively. See Figure 6 for monomer structures. Random copolymers show expected cyclic voltamograms with distinct two redox peaks at 30 mV and 330 mV (versus Fc/Fc ) ( Figure 10B). Ratios of peak areas are 4.7:1 and X/X, respectively. These results demonstrate redox potentials and current ratio can be utilized as versatile indicators for multi channel DNA detection.
  • the polymer (poly2-5-7, 125 mg, 82% mimmum isolated yield) was isolated by adding the mixture dropwise into a stined solution of pentane, filtering, and repeatedly washing with fresh pentane (4 x 20 mL).
  • composition In an inert atmosphere glovebox, 2 (70 mg, 0.22 mmol), 5 (58mg, 0.125 mmol), and 7 (27mg, 0.063 mmol) were weighed into a 25-mL round bottom flask equipped with a magnetic stirring bar. Dry THF (3 mL) was added, followed by a solution of catalyst 1 (10.3mg, 0.013mmol) in dry THF (0.5 mL). The mixture was stined for 10 h, the reaction was removed from the dry box and the polymerization was terminated with ethyl vinyl ether (1 mL).
  • the polymer (poly2-5-7, 100 mg, 65% mimmum isolated yield) was isolated by adding the mixture dropwise into a stined solution of pentane, filtering, and repeatedly washing with fresh pentane (4 x 20 mL).
  • Poly2-5-7 (55 mg of the (17:10:5) composition) was dissolved in dry THF (2 mL). Diisopropylethylamine (100 ⁇ L) and 3 (70 mg, 0.296 mol) were added and the mixture was stined at room temperature for 4 h. The mixture was poured into pentane (100 mL), filtered, washed with fresh pentane (4 x 20 mL), dried over sodium sulfate, and concentrated to dryness to yield the desired product which was dissolved in CDCI3 for P NMR analysis and then used directly in the next step. 31 P NMR (CDCI3): ⁇ 149.2.
  • UN-Nis spectra of the aggregates showed significantly reduced D ⁇ A signal at 260 nm and increased intensity at higher wavelength due to scattering from micrometer size polymer aggregates, Figure 11 A.
  • blockcopolymers were redispersed in solution evidenced by UN-Nis spectrum, Figure 11 A.
  • the thermal denaturation curves of these aggregates were obtained by monitoring the UN-Nis spectra at 260 nm as a function of temperature, Figure 11B.
  • the aggregates formed from D ⁇ A blockcopolymer show higher thermal stability and extraordinarily sha ⁇ er melting transition than plain duplex D ⁇ A.
  • a melting curve of D ⁇ A duplex with same D ⁇ A sequences as Hybrid V:VI are presented in Figure 1 IB for comparison.
  • the melting temperature of aggregates formed from hybrid molecules is higher than D ⁇ A duplexes by 14 °C.
  • the higher thermal stability has been observed in D ⁇ A dendrimers, and is consistent with multiple linkage and cooperative effect. This property is important for detecting double strand D ⁇ A.
  • the half maximum full widths (HMFW) of the derivatives of melting curve of hybrid molecules are 2 °C ( Figure 1 IB, inset).
  • the degree of sha ⁇ melting transition has been observed in oligonucleotide-modified particles but not in DNA dendrimers.
  • the particle based detection methods also show high selectivity due to the sha ⁇ , melting characteristics.
  • Example 8 DNA Detection Using Hybrid Molecules.
  • DNA-modified ROMP blockcopolymers with various redox potentials can be readily prepared from norbomenes substituted with electrochemical tags. These polymers showed fully active DNA recognition properties and expected electrochemical properties. Importantly, DNA blockcopolymers exhibit useful properties such as sha ⁇ melting transitions and high thermal stabilities. This strategy can be extended to prepare virtually any other norbornene monomers, thereby imparting unprecedented functionality to branched DNA structures.

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Abstract

La présente invention concerne des polymères ou copolymères à polymérisation méthatétique par ouverture des noyaux, c'est-à-dire de type ROMP (Ring-Opening Metathesis Polymerization), auxquels sont liés des oligonucléotides. L'invention concerne également des matériaux faits de ces polymères ROMP à modification oligonucléotidique. L'invention concerne enfin des procédés de fabrication et d'utilisation de ces produits pour péparer de nouveaux matériaux et pour la détection d'acides nucméiques cibles.
PCT/US2002/012071 2001-04-26 2002-04-18 Polymers et copolymeres romp a modification oligonucleotidique WO2003087188A1 (fr)

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AU2002367709A AU2002367709A1 (en) 2001-04-26 2002-04-18 Oligonucleotide-modified romp polymers and co-polymers

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US8323888B2 (en) 1996-07-29 2012-12-04 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
US7985539B2 (en) 2002-05-07 2011-07-26 Northwestern University Nanoparticle probes with raman spectroscopic fingerprints for analyte detection
GB2423987A (en) * 2005-03-09 2006-09-13 E2V Tech Metallocenes and their use as Raman spectroscopy labels
US9662403B2 (en) 2008-05-13 2017-05-30 University Of Washington Micellic assemblies
US11707483B2 (en) 2008-05-13 2023-07-25 University Of Washington Micellic assemblies
US9006193B2 (en) 2008-05-13 2015-04-14 University Of Washington Polymeric carrier
US9339558B2 (en) 2008-05-13 2016-05-17 University Of Washington Micellic assemblies
US10420790B2 (en) 2008-05-13 2019-09-24 University Of Washington Micellic assemblies
US9862792B2 (en) 2008-05-13 2018-01-09 University Of Washington Diblock copolymers and polynucleotide complexes thereof for delivery into cells
US9476063B2 (en) 2008-05-13 2016-10-25 University Of Washington Diblock copolymers and polynucleotide complexes thereof for delivery into cells
US9211250B2 (en) 2008-08-22 2015-12-15 University Of Washington Heterogeneous polymeric micelles for intracellular delivery
US9220791B2 (en) 2008-11-06 2015-12-29 University Of Washington Bispecific intracellular delivery vehicles
US9464300B2 (en) 2008-11-06 2016-10-11 University Of Washington Multiblock copolymers
US8822213B2 (en) 2008-11-06 2014-09-02 University Of Washington Bispecific intracellular delivery vehicles
US9593169B2 (en) 2008-12-08 2017-03-14 University Of Washington Omega-functionalized polymers, junction-functionalized block copolymers, polymer bioconjugates, and radical chain extension polymerization
US10066043B2 (en) 2008-12-08 2018-09-04 University Of Washington ω-functionalized polymers, junction-functionalized block copolymers, polymer bioconjugates, and radical chain extension polymerization
US9415113B2 (en) 2009-11-18 2016-08-16 University Of Washington Targeting monomers and polymers having targeting blocks
US9867885B2 (en) 2013-07-30 2018-01-16 Phaserx, Inc. Block copolymers
US10646582B2 (en) 2013-07-30 2020-05-12 Genevant Sciences Gmbh Block copolymers
US10660970B2 (en) 2013-07-30 2020-05-26 Genevant Sciences Gmbh Nucleic acid constructs and methods of using the same
US11938191B2 (en) 2013-07-30 2024-03-26 Genevant Sciences Gmbh Block copolymers
US11219634B2 (en) 2015-01-21 2022-01-11 Genevant Sciences Gmbh Methods, compositions, and systems for delivering therapeutic and diagnostic agents into cells
US11684584B2 (en) 2016-12-30 2023-06-27 Genevant Sciences Gmbh Branched peg molecules and related compositions and methods
US10973927B2 (en) 2017-08-28 2021-04-13 The Chinese University Of Hong Kong Materials and methods for effective in vivo delivery of DNA nanostructures to atherosclerotic plaques

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