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WO1993006122A1 - Conjugues polynucleotidiques a formation de doubles helices - Google Patents

Conjugues polynucleotidiques a formation de doubles helices Download PDF

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Publication number
WO1993006122A1
WO1993006122A1 PCT/CA1992/000423 CA9200423W WO9306122A1 WO 1993006122 A1 WO1993006122 A1 WO 1993006122A1 CA 9200423 W CA9200423 W CA 9200423W WO 9306122 A1 WO9306122 A1 WO 9306122A1
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Prior art keywords
polynucleotide
duplex
forming
terminus
duplexed
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PCT/CA1992/000423
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English (en)
Inventor
Michael Y.-X. Ma
Lorne S. Reid
Martin Sumner-Smith
Richard W. Barnett
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Allelix Biopharmaceuticals Inc.
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Publication of WO1993006122A1 publication Critical patent/WO1993006122A1/fr

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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • This invention is in the field of nucleic acid chemistry. More particularly, the invention relates to polynucleotide conjugates that adopt a ligand binding duplexed structure, to the production of such conjugates particularly via automated synthesis techniques, and to their use in therapeutic, diagnostic and other applications.
  • oligonucleotide-based pharmaceuticals are designed according to their nucleic acid sequence to arrest genetic processes by binding disruptively to a selected genetic target, usually a viral gene or a human gene that is associated with a particular disease state such as cancer or a condition such as inflammation.
  • Transcription of an undesired gene can, for example, be arrested by a synthetic oligonucleotide that hybridizes selectively to a control region or coding region of that gene; similarly, translation of an undesired protein can be arrested using an oligonucleotide that hybridizes with a control region or coding region of the messenger RNA encoding that protein.
  • oligonucleotide-based therapeutics such as cell uptake, stability, and cost of production, have been resolved by recent advances in nucleic acid chemistry.
  • oligonucleotides which, in order to hybridize to their intended nucleic acid target, are necessarily single-stranded complements of that target. That is, oligonucleotides intended for use as pharmaceuticals are designed currently to bind as single-stranded entities to other nucleic acid targets, whether single-stranded messenger RNA or single stranded DNA (the so- called “sense” and “anti-sense” approaches, reviewed for example by Uhlmann et al, 1990, Chemical Rev., 90:543) or, as has more recently been proposed, to double stranded DNA (the "triplex” approach). These approaches neglect other cellular targets that are at least equally attractive in the overall development of gene regulating therapeutics. More particularly, it would be desirable to provide oligonucleotide agents capable of interfering with interactions specifically between nucleic acids and their ligands, particularly their protein ligands, having a role in infectious and other disease states.
  • oligonucleotides that interfere with a protein/nucleic acid interaction of therapeutic interest is complicated in that, in the majority of instances, the protein recognizes a nucleic acid that is double stranded in structure; and further in that double stranded oligonucleotides of the small size necessary for pharmaceutical applications, for uptake by the cell, and for stability, are highly unstable and must typically be incubated under temperatures so cold and/or salt concentrations so high as to make subsequent study and use of the duplexed structures impractical.
  • polynucleotide conjugates which comprise a first polynucleotide strand having an end, a second polynucleotide strand having an end and which is capable of annealing with the first polynucleotide strand to form a ligand binding structure, and a chemical linker which is coupled between ends of the strands to form a bridge permitting the conjugate to form a ligand-binding duplexed structure.
  • the stability- enhanced duplexed structures of the invention are provided in the form of linear polynucleotide conjugates, conforming to the general formula:
  • X is a polynucleotide having a 3'terminus
  • Y is a polynucleotide capable of annealing with X, and having a 5'terminus
  • L is a chemical linker coupled between the 3'terminus of X and the 5'terminus of Y to form a bridge permitting the conjugate to form a ligand-binding duplexed structure.
  • Compounds conforming to the general formula (I) are linear polynucleotide conjugates and are most conveniently produced using automated polynucleotide synthesis techniques.
  • the pre&ant invention further provides analogues of the chemical linkers in bifunctional form for incorporation between nucleotide strands using established nucleotide coupling protocols.
  • the stability-enhanced duplexed structures may also be in the form of cyclic polynucleotide conjugates, which conform either to the general formula:
  • X is a polynucleotide having a 5'terminus and a 3'terminus
  • Y is a polynucleotide capable of annealing with X and having a 3'terminus and a 5'terminus;
  • Z is a polynucleotide coupled covalently between the
  • L is a chemical linker coupled between the 3'terminus of X and the 5'terminus of Y, to form a bridge permitting the conjugate to form a ligand-binding duplexed structure;
  • LI and L2 are independently selected chemical linkers coupled, respectively, between the 3'terminus of X and the 5'terminus of Y and the 5'terminus of X and the 3'terminus of Y, to form chemical bridges permitting the conjugate to form a ligand-binding duplexed structure.
  • cyclic polynucleotide conjugates of the invention as represented by formulae Ha and lib are suitably prepared by synthesizing the linear analogue thereof using the automated nucleotide coupling techniques appropriate for linear conjugates of formula (1) and then closing the linear conjugate typically using either chemical or enzymatic means, to form the cyclic polynucleotide conjugate.
  • a pharmaceutical composition which comprises a ligand- binding polynucleotide conjugate of the present invention and a pharmaceutically acceptable carrier.
  • the polynucleotide conjugate is one capable of adopting a duplexed structure that is recognized by i.e. binds with, a target ligand that is a protein, for example a protein capable of regulating gene expression.
  • the polynucleotide conjugate is characterized by an affinity for binding with protein which regulates viral gene expression e.g. the HIV tat protein.
  • the polynucleotide conjugate can be designed by appropriate selection of its component polynucleotide strands and linker(s) to bind with proteins that regulate oncogene expression, or expression of genes implicated in other disease states or medical conditions.
  • the chemical linker component of the polynucleotide conjugate incorporates a functional group which serves as a site of attachment for a reporter molecule, such as a radiolabel or other diagnostically useful label.
  • the invention further provides detectably labelled analogues of the polynucleotide conjugates, for diagnostic use or for use in assays
  • UBSTITUTE SHEET designed to measure binding between the duplexed form of the conjugate and a ligand, such as a DNA- or RNA-binding protein. Further, the attachment site within the chemical linker may be exploited to couple the polynucleotide conjugate to an affinity column matrix, for use in extracting ligands from biological sources.
  • Figures 1 and 2 illustrate duplexed structures of various conformations and configurations that can be stabilized in accordance with the present invention.
  • Solid lines illustrate polynucleotide structure and hatching identifies the nucleotide components.
  • the symbol ".” is used to indicate hydrogen-bonded base-pairing within annealed regions of the polynucleotide strands, and the symbol L is used to indicate location of the chemical linker;
  • Figure 3 shows incorporation of a specific linker of the present invention between polynucleotide strands
  • FIGS 4-7 illustrate the structure of specific polynucleotide conjugates of the present invention.
  • Figures 8 and 9 illustrate graphically the cellular uptake of specific polynucleotide conjugates of the invention.
  • the present invention provides polynucleotide conjugates characterized by the properties of ligand binding and enhanced stability.
  • the term “enhanced stability” refers unless otherwise stated to the superior thermal stability of a polynucleotide conjugate relative to its unlinked counterpart, as measured using melting temperature (Tm) assays established in the art.
  • Tm melting temperature
  • ligand is used herein with reference to agents that bind measurably, in the context of an assay appropriate for that measurment, to nucleic acid structures, principally double stranded structures but also single stranded structures.
  • the term ligand is thus intended to embrace such agents as proteins, including proteins that regulate genetic processes such as transcription and translation, as well as non-protein entities including but not limited to intercalating agents and nucleic acid binding antibiotics as well as other nucleic acids.
  • ligand-binding is thus used with reference to polynucleotide conjugates that adopt a structure that is bound measurably by a ligand to which the conjugate is targetted.
  • the present invention permits the use of double stranded polynucleotide structures in a wide variety of applications not previously possible, because of prior stability problems. Because the chemically linked duplexed structures of the present invention are substantially more stable than their unlinked counterparts under physiological conditions, for example, therapeutic applications for duplexed structures are now feasible. In addition, it will be appreciated that the stability-enhancing effect of the chemical linker can be exploited to eliminate polynucleotide regions that are otherwise required to permit formation and maintenance of the desired duplexed structure in vitro and in vivo. Thus, duplexed structures that are much smaller in molecular weight and accordingly more acceptable for therapeutic use, can be produced. Furthermore, the chemical linkers exploited in the present invention are substantially resistant to nuciease digestion, and thus further contribute to duplex stability.
  • the present invention applies the strategy of incorporating a chemical linker between one or both ends of polynucleotide strands capable of forming a duplexed structure. It will be understood that in order to form a duplexed structure, such strands will share at least a region of sequence complementary sufficient to permit annealing of the strands.
  • the individual polynucleotide strands forming the duplex may consist of RNA or DNA monophosphates or synthetic analogues thereof, or mixtures thereof.
  • Synthetic analogues include for example those incorporating variations in the base constituent, such as thio- and aza-substituted bases; in the sugar consitituent such as alkyl- and halo-substituted riboses and arabinose equivalents; and analogues incorporating variation in the monophosphate group, such as phosphorothioates and dithioates, methyl phosphate and methyl phosphonates, phosphoramidates and phosphoramidites and the like.
  • a polynucleotide strand may also incorporate a non- nucleic acid component, to the extent that duplex formation and ligand binding are not substantially impaired.
  • the polynucleotide strands forming the duplex may be of the same or different lengths, and each may incorporate any number of nucleotides in the range from 2 to a maximum that is dictated largely by the limits of automated gene synthesis techniques. Strands consisting of not more than about 200 nucleotides, for example not more than about 100 nucleotides, will derive the most benefit from the stabilizing effect of the chemical bridge, however. Preferably each of the polynucleotide strands consists of from 3 to 100 nucleotides, and more preferably, from about 4 to 50 nucleotides.
  • Polynucleotide strands that are capable of annealing, and which can thus benefit from the linker strategy herein described, include those strands that anneal in their anti-parallel orientation i.e. consist of beta nucleotides, and strands that consist of alpha nucleotides in one strand and beta nucleotides in the other strand, and thus can anneal in the parallel orientation.
  • the polynucleotide strands that are capable of annealing, and which can thus benefit from the linker strategy herein described include those strands that anneal in their anti-parallel orientation i.e. consist of beta nucleotides, and strands that consist of alpha nucleotides in one strand and beta nucleotides in the other strand, and thus can anneal in the parallel orientation.
  • SUBSTITUTE SHEET strands will be precisely complementary and equivalent in length, and will anneal along their entire length, to form a completely double stranded duplexed structure. It will be appreciated however, that with the aid of a chemical linker, duplexed structures having a variety of conformations and configurations can be stabilized, in accordance with the present invention. Some of the duplexed structures currently contemplated are illustrated schematically in Figures 1 and 2, to which reference is now made. Other structures or combinations may also be stabilized in accordance with the present invention, of course.
  • duplexed structures that can be generated as linear polynucleotide conjugates of the general formula (I) comprise a single chemical linker incorporated at one end of the duplex structure.
  • Figure 1 (a) illustrates the simplest case which, as described above, incorporates a linker at one end of precisely complementary polynucleotide strands, which anneal along their entire length to form a fully double stranded duplex structure.
  • Figure 1 (b) illustrates the case in which the annealable strands incorporate a terminal mismatch, which results in a non-annealing "fork" structure at one end of the duplex.
  • Figure 1 (c) illustrates the situation in which one polynucleotide strand incorporates an internal, mismatched region resulting in a non-annealed bulge.
  • Figure 1 (c) further illustrates that polynucleotide strands of different length can also be linked, according to the present invention, as is further shown by the structure of Figure 1 (d).
  • duplexed structures that can be generated as cyclic polynucleotide conjugates of the formula (Ha) and (lib) may also adopt various conformations and configurations.
  • Figure 2(a) the simplest case is again the situation where precisely complementary strands are coupled using chemical linkers at both ends.
  • the forked structure shown in Figure 2(b) can also be linked at both ends, as may the bulged structure shown in Figure 2(c).
  • SUBSTITUTE SHEET Figure 2(b) also illustrates that chemical linkers of different length may be used to bridge polynucleotide strands in the annealing relationship desired for duplex formation.
  • Duplexes that are more elaborate in structure can also be stabilized if desired, as shown for example in Figures 2(d) and 2(e).
  • the duplexed structures appearing in Figures 2(a) - (e) are intended to be embraced by the general formula 1Kb) recited hereinabove.
  • the duplexed structure illustrated in Figure 2(f) represents a special but important case, in which a cyclic duplexed structure is created by incorporation of a single chemical linker, as embraced generally by the formula II (a) recited hereinabove.
  • Z is represented by the polynucleotide 'loop' bridging the annealed polynucleotide strands.
  • such structures exist naturally in the unlinked form, occurring predominantly in the form of RNA "hairpins" that regulate the expression of certain viral and other genes through a protein-binding interaction.
  • Such duplexed structures are accordingly ideal as targets for therapeutic application, when in their chemically linked form.
  • the linking of duplex-forming polynucleotide strands is achieved by covalently coupling the chemical linker between neighbouring termini of the polynucleotide strands, either between the ⁇ terminus of one strand and the 3'terminus of the other, or vice versa.
  • linkers are most suitably incorporated by coupling between the monophosphate or analogous groups borne at the termini.
  • the chemical linkers used in the present invention are synthetic chemical linkers as opposed to polynucleotide-based linkers of the type represented by substituent Z in Formula (lla).
  • the chemical linker has a length selected ideally to preserve the desired annealing relationship between strands at the location of the linker. Since numerous duplex conformations can be stabilized using the
  • linkers of similarly various lengths can be incorporated for this purpose.
  • the length of the linker will correspond to the length of a linear chain alkane comprising from about three carbon atoms (C 3 ) to about 30 carbon atoms (C 30 ).
  • C 3 carbon atoms
  • C 30 carbon atoms
  • a chemical linker having a length equivalent to a linear chain alkane consisting of from 7 to 20 carbon atoms, suitably 8 to 15 carbon atoms and desirably 9-12 carbon atoms is appropriate to link polynucleotide strands at an annealed location.
  • a chemical linker having a length equivalent to greater than about 10 carbon atoms, for example having a length in the range from about 10 carbon atoms to about 20 carbon atoms, is suitable for incorporation. Since functional groups are also incorporated at the ends of the linker to permit coupling with nucleotides, as described below, determination of desired linker length should be made with this in mind.
  • the chemical composition of the linker can vary widely, provided that consideration is given to the need for stability under physiological conditions and under the conditions encountered during nucleotide coupling protocols.
  • the linker may contain functional groups, for example to serve as attachment sites for other molecular entities, provided that suitable protecting groups are employed during synthesis of the polynucleotide conjugate.
  • a key requirement in choosing a linker composition is to retain the length appropriate for duplex formation.
  • side chains are acceptable, particularly in the central region of the linker.
  • the desired length of the linker can be achieved using carbon atoms or carbon atoms in combination with heteroatoms, including oxygen, sulfur, phosphorus, nitrogen, etc.
  • cyclic structures can be incorporated, including benzene and heterocycles such as piperidine, piperazine or pyridine coupled within the linker chain either through a carbon center or a heteroatom. It will also be appreciated that the chemical composition of
  • linker can be manipulated through component selection to alter hydrophobicity or hydrophilicity, if desired, particularly for the purpose of altering solubility, cellular uptake, and to facilitate dosage formulation where therapeutic applications are being considered.
  • the chemical linkers are provided in the form of bifunctional analogues, bearing terminal functional groups that, desirably, are amenable to protection and derivatization that adapts them for coupling using the same protocols applied conventionally for automated nucleotide coupling.
  • bifunctional linker analogues conform to the general formula,
  • R and R' are independently selected from among the group consisting of -OH, -SH, -NH and functional equivalents of these groups.
  • the linker is preferably one in which at least one of R and R' is OH. Most preferably, both R and R' are OH.
  • Bifunctional linkers suitable for use in coupling polynucleotide strands at an annealed location are exemplified by, and include:
  • linkers of appropriate length may also be formed in situ i.e. during conjugate synthesis, by coupling selected linkers sequentially to extend linker length as desired.
  • polynucleotide conjugates of general formula (I), which are linear molecules capable of forming duplexed structures can be synthesized by applying now conventional techniques of polynucleotide synthesis, particularly in combination with the commercially available polynucleotide synthesizing devices, or "gene machines".
  • Various strategies of solution and solid phase synthesis can be used, of course, including the phosphotriester method, the solid phase H-phosphonate method or the solid phase phosphoramidite method. The latter is currently the method of choice, for synthesis of polynucleotide-based compounds of the invention.
  • nucleotides that are fully protected are coupled sequentially, in the 3' --> 5' direction, to a first nucleotide that is coupled releasably to a solid support, such as aminopropyl controlled pore glass or polystyrene resin.
  • Nucleotide protecting groups include, for nucleophilic amino functions on the bases, either isobutyryl (N-2 of guanine) or benzoyl (N-6 of adenine and N-4 of cytidine) that are removable upon completion of synthesis by ammoniolysis.
  • the 5' primary hydroxyl of the deoxyribose sugar is protected with an ether moiety, either dimethoxytrityl (DMT) or monomethoxytrityl (MMT), which is removed by mild protic acids at the beginning of each coupling cycle.
  • DMT dimethoxytrityl
  • MMT monomethoxytrityl
  • the 3' secondary hydroxyl function of the deoxyribose sugar is derivatized with the highly reactive phosphoramidite group, either methyl phosphoramidite or ⁇ -cyanoethyl phosphoramidite, which is activated for coupling by a weak acid.
  • the bifunctional linker analogues of the present invention can be similarly protected and deprotected for coupling.
  • the linker analogue bears terminal hydroxyl groups
  • these may be protected in the same manner as the 5' and 3' hydroxyls of the nucleotides selected for coupling.
  • one hydroxyl is protected with the ether moiety, such as DMT, and the other is derivatized to provide the phosphoramidite group, to yield a compound of the general structure, DMT-O-linker-O-phosphoramidite.
  • DMT-O-linker-O-phosphoramidite This permits unidirectional incorporation of the linker into the linear polynucleotide, at a desired position along its length.
  • Example 1 Techniques for obtaining linkers suitably adapted for nucleotide coupling reaction are provided in Example 1 herein. Briefly, for dimethoxy or monomethoxy tritylation, the trityl halide and a slight molar excess of the diol are reacted in pyridine at room temperature, and the product is recovered after mixing with methanol, resuspension in chloroform and then washing and drying, with solvent removal. The tritylated product can then be phosphitylated, to protect the remaining hydroxyl group, by reaction with 2-cyanoethyl-N,N-diisopropylchorophosphoramidite in the conventional manner. The so-protected diol linker can then be incorporated into an automated nucleotide synthesis protocol in the same manner as would any protected nucleotide.
  • the resin-bound first nucleotide is treated with protic acid to remove the trityl protecting group at the 5'hydroxyl, the 3'hydroxyl phosphoramidite group of the next nucleotide is activated to allow 3' to 5' coupling, and then oxidized to complete coupling.
  • the protected linker is incorporated using the same deprotection/activation strategy and the coupling continues until the linear form of the double stranded oligonucleotide is produced. This is then released from the solid support
  • Figure 3 provides the chemical structure resulting from the covalen coupling of a specific triethylene glycol-derived linker, between polynucleotides. It will be noted that the linker is coupled to the termini of the nucleotides through the monophosphates borne on the respective 5' and 3' hydroxyl groups.
  • RNA-based, linear polynucleotide conjugates can be employed for synthesis of RNA-based, linear polynucleotide conjugates, but with use of a blocking group for the 2'hydroxyl, such as the tert-butyldimethylsilyl group (TBDMS) or the triisopropylsilyl group (TIPS), and optionally with use of the MMT or DMT ethers for 5'hydroxyl protection.
  • TDMS tert-butyldimethylsilyl group
  • TIPS triisopropylsilyl group
  • a linear analogue of the cyclic molecule is first produced using the procedure described above for linear polynucleotide conjugate production.
  • the linear analogue is produced such that the ends of the resulting linear conjugate can be closed either by chemical reaction or by enzymatic ligation. Chemical closure can be achieved using various available techniques.
  • One convenient approach requires fully-deprotected linear precursor sequences and use of chemical condensation reagents, such as cyanogen bromide as described by Prakash, G. et al (1992) J. Am. Chem.
  • the typical condensation reagent in this case is 1 -(2-Mesitylenesulfonyl)- 3-Nitro-1 ,2,4-TriazoIe (MSNT).
  • MSNT 1-Mesitylenesulfonyl
  • the cyclic oligonucleotides are treated according to standard procedures of deprotection and purification.
  • Another alternative approach generates a fully-protected cyclic oligonucleotide directly on the polymer-support (see Barbato, S. et al., (1989) Tetrahedron, 45:4523; and Capobianco, M.L. et a/., (1990) Nucleic Acids Res. ,18:2661 ).
  • This phosphotriester approach does not require a post-synthesis cyclization, and results cyclic molecules with high efficiency.
  • Cyclization of the polynucleotide conjugate can also be achieved by enzymatic ligation of the free ends of a linear conjugate.
  • the ends to be ligated correspond preferably to an annealing site in the duplexed structure, to facilitate action of the enzyme, preferably an RNA or DNA ligase, as appropriate.
  • the linear conjugate is preferably incubated first under annealing conditions and then treated with either RNA or DNA ligase.
  • RNA ends can be annealed in similar fashion, by treatment with RNA ligase in particular.
  • the cyclic polynucleotide conjugates resulting from the reaction can be recovered and purified using techniques established generally for polynucleotides, and as described in the examples herein.
  • the polynucleotide strands to be linked during synthesis are selected in terms of their nucleic acid sequence, and based on knowledge of the particular nucleic acid sequence to which a target ligand binds. It will be appreciated that selection of strands appropriate for desired ligand binding can be guided by the vast scientific literature dealing with protein/nucleic acid interactions. In those instances where a binding domain of specific interest remains to be identified, it will be appreciated that the mapping of that domain can be achieved using
  • SUBSTITUT conventional approaches, so that a specific binding sequence can be elucidated.
  • the strategy hereindescribed can in fact facilitate such mapping, by permitting the synthesis of a series of stabilized duplexed structures representing putative ligand binding domains that can then be screened for ligand binding activity using for example the mobility shift assays of the type hereindescribed.
  • the polynucleotide conjugates are employed as mimics of naturally occurring duplexed structures, and the polynucleotide strands in the conjugate are accordingly selected to correspond in sequence to a naturally occurring duplex counterpart.
  • any duplexed region of a naturally occurring gene or other genetic element can be duplicated in stability- enhanced form, in accordance with the present invention.
  • Ligands of potential interest include those proteins which on binding to their natural, nucleic acid target, directly or indirectly, influence the utilization or fate of that nucleic acid target.
  • proteins include: ribo- and deoxyribonucleoprotein complexes; gene regulatory proteins such as repressors, activators and transactivators, etc.; proteins involved in the modifications and fate of mRNA molecules, including splicing, polyadenylation, capping, nuclear export, translation, degradation, etc.; proteins involved in the assembly and utilization of other RNA or ribonucleoprotein structures such as ribozymes, tRNA synthetases, splicing complexes, etc.
  • the essential feature of such proteins is that they recognise particular nucleic acid structures on the basis of their conformation and/or sequences; embodiments of this invention would provide effective analogues when they maintain some or all of such requirements.
  • polynucleotide conjugates that bind ligands other than protein ligands, e.g. chemical ligands such as intercalating
  • SUBSTITUTE SHEET agents e.g. psoraien and ethidium bromide
  • nucleic acid-binding antibiotics e.g. distamycin and netropsin
  • other nucleic acid structures e.g. psoraien and ethidium bromide
  • the polynucleotide conjugates comprise polynucleotide strands which, in their duplexed form, exhibit binding affinity for the tat protein of the human immunodeficiency virus, HIV.
  • the tat protein mediates a rapid increase in the production of the viral components required for HIV replication, which in turn leads to the onset of AIDS. It has been suggested that agents capable of interfering with the tat/Tar interaction will be useful in arresting HIV replication, and thus efficacious in the treatment of AIDS.
  • the present invention accordingly provides a polynucleotide conjugate which is capable of adopting a duplexed structure having a binding affinity for tat.
  • the polynucleotide conjugate has a chemical structure described in the examples herein. It will be appreciated, however, that sequence variation can be tolerated without loss of tat binding affinity, and such variations which retain tat binding are within the scope of this embodiment of the present invention.
  • duplexed RRE RNA structure required to regulate splicing and the duplexed tRNA Ly ⁇ 3 structure used as a primer for reverse transcription can be mimicked using the present strategy.
  • duplexed structures which bind other regulatory protein ligands, for example those known to exist in human pathogenic viruses, including: the P protein of Hepatitis B
  • HSV SUBSTITUTE SHEET virus
  • HPV Papilloma virus
  • BZLF1 and EBNA-1 proteins of Epstein Barr virus (EBV) as well as additional proteins in these and other viruses.
  • HSE heat shock element
  • Formulation and administration of the compounds herein described, and indeed any annealed polynucleotide structures having pharmaceutical utility, can be accomplished in accordance with procedures routinely applied to aqueous-soluble compounds.
  • buffered saline solutions are acceptable.
  • timed-release polymeric compositions which do not unfavourably chemically modify the compounds are acceptable. Modification of pharmacokinetic properties, especially distribution, are achieved, for instance, through the use of iiposomal or cationic lipid formulations.
  • the polynucleotide conjugates comprise polynucleotide strands which in their duplexed form present nucleic acid epitopes of interest, for example as immunogens suitable for raising antibodies.
  • the raising of such antibodies can be achieved in the manner conventional for polyclonal antibody production, or for monoclonal antibody production.
  • Such antibodies will find utility in assays designed to detect eptiopes against
  • SUBSTITUTE SHEET which the antibodies were raised, especially when conjugated to a suitable reporter molecule; and may also be useful in protecting a region of a polynucleotide while manipulating that polynucleotide at another site.
  • the polynucleotide conjugate may be coupled via an attachment site incorporated within the chemical linker, to a desired agent such as a cross-linking agent, reporter molecule, cell uptake enhancer such as lipid or cholesterol, alkylating groups, chromatographic beads and other functional groups .
  • a desired agent such as a cross-linking agent, reporter molecule, cell uptake enhancer such as lipid or cholesterol, alkylating groups, chromatographic beads and other functional groups .
  • a variety of chemical groups may serve as attachment sites, provided of course that such groups permit coupling of the polynucleotide strands as desired, and can be protected during polynucleotide synthesis.
  • the attachment site is constituted by a chemical entity that can be protected by a base-labile protecting group removable by ammoniolysis.
  • One such group is the FMOC group used in conventional peptide synthesis protocols.
  • the attachment site may be constituted by a phosphate group incorporated within the linker, which can be derivatized following oxidation from either H-phosphonate to phosphate triester, or phosphite triester to phosphate triester.
  • the conjugates of the invention can may also be used diagnostically e.g. as a competing ligand, to assay specimens for the presence of target ligand in a qualitative or semi-quantitative fashion, for example using a competitive binding assay format.
  • a reporter such as a radiolabel
  • ICl DMTr-O-(CH 2 ) 3 -OH, yield 67.0% from 1 ,3-propanediol [1 .7 g (22.5 mmol)]; DMTr-CI [5.0 g (15 mmol)]; and pyridine [100 ml].
  • Example 2 Phosphitylation of tritylated linkers
  • the tritylated linker prepared as described in Example 1 was next derivatized at the remaining hydroxyl group to incorporate a phosphoramidite group, according to the reaction scheme illustrated below:
  • reaction mixture was stirred at room temperature for 1-2 hours and monitored by TLC (EtOAc/CH 2 CI 2 /TEA, 45:45:10, v/v).
  • Linker A DMT-O-(CH 2 ) 9 -O-phosphoramidite
  • Linker B DMT-O-(CH 2 CH 2 O) 2 -CH 2 CH 2 -O-phosphoramidite TLC (silica gel, petroleum ether/EtOAc/TEA, 50:10:1 , v/v/v): R f
  • Linker D DMT-O-(CH 2 CH 2 O) 5 -CH 2 CH 2 -O-phosphoramidite
  • Controlled pore glass was used as the solid support matrix for both DNA & RNA synthesis.
  • Polvdeoxyribonucleotides (DNA) were prepared by the CE-phosphoramidite method on an Applied Biosystems 391 EP synthesizer (0.15 micromole scale). . Cleavage and deprotection were effected by standard ammonia treatment.
  • Oligoribonucleotides (RNA) were prepared according to the method of Usman et al, 1987, J. Am. Chem.
  • Soc, 109, 7845-7854 employing 5'-dimethoxytrityl-2'-t- butyldimethoxysilyl ribonucleoside-3'-CE- phosphoramidites (Peninsula Labs, CA or ChemGenes Corp., MA). Syntheses were carried out on an Applied Biosystems 380B synthesizer using a modified 0.2 micromole cycle. Cleavage from the support, base & phosphate deprotection, and removal of the 2'-TBDMS groups were performed by established procedures (Scaringe et al, 1990, Nucl. Acids Res., 18, 5433-5441 ). The crude oligonucleotide in TBAF solution was desalted on a C 18 Sep-Pak cartridge prior to purification.
  • the linker phosphoramidite (dissolved in dry acetonitrile, 0.2-0.3 M) was coupled to the support-bound polynucleotide at the desired location, using the synthesis cycle conventional for standard nucleoside phosphoramidites.
  • RNA oligomers were further characterized by enzymatic RNA sequencing [Donis-Keller, H. (1980) Nucleic Acids Res., 8, 3133-3142] or base-composition analysis [Seela, F. & Kaiser, K. (1987) Nucleic Acids Res., 15, 31 13-3129].
  • linear polynucleotide conjugate 2 which contains triethylene glycol-derived linker B, having the structure -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O; and linear polynucleotide conjugate __. which contains propanediol-derived linker C, having the structure -O- (CH 2 ) 3 -O-. If the length and nature of linker B has been selected appropriately, polynucleotide conjugate 2 should adopt a duplexed structure that is digested more rapidly by EcoRI than the unlinked control molecule 1. The linker C in conjugate _! is expected to be too short to permit functional annealling of the strands, which should translate into slower EcoRI digestion relative to conjugate 2.
  • the conjugates were
  • oligonucleotides (5 pmol) were dissolved in 70 mM Tris-HCl (pH 7.6), 10 mM MgCI 2 , 10 mM KCI and 5 mM dithiothreitol (DTT) and incubated with 9 pmol of - 32 P-labelled ATP and 10 units of T4 polynucleotide kinase (New England Biolabs) at 37°C for 1-2 h. The reaction was terminated by heating the mixture to 80°C for 10 min, and then was slowly cooled to room temperature.
  • Tris-HCl pH 7.6
  • DTT dithiothreitol
  • the solution was desalted by passage through a Bio-spin column (BIO-RAD, Bio-spin 6 for the unlinked control, and Bio-spin 30 for the polynucleotide conjugates).
  • An alternative method for purification of labelled polynucleotide involved one ⁇ time extraction with an equal volume of phenol solution and precipitation using two volumes of ethanol/acetate (1 :1 ; v/v) at -20°C overnight, with collection and drying under high speed vacuum.
  • EcoRI digestion 1 pmol of the selected, 32 P-labelled substrate was incubated with 20 units of EcoRI (Pharmacia) in 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCI 2 , 1 mM 0ME and 100 /g BSA/ml (total volume: 20 ⁇ l). The reactions were carried out at room temperature, and 2 ⁇ of sample was removed at different time intervals. The samples were analyzed on a 20% denaturing polyacrylamide gel.
  • SUBSTITUTE SHEET enhance the stability of and retain the function of duplexed structures, including those having protein binding affinity.
  • RNA structure known as Tar consists of 59 bases in most HIV-
  • linear polynucleotide conjugates representing analogues of a 27-mer truncated version of Tar ( Figure 4) were synthesized and evaluated. All were prepared using the synthesis procedures previously described hereinabove. As Figure 4 illustrates, the linear polynucleotide conjugates tested comprised two classes; one class in which the 6-mer loop in the Tar analogue (4) was replaced by each of four different linkers (conjugates 5A, 5B, 5C and 5D) and another class in which the 6-mer loop was replaced by two coupled linkers (5BB and 5CC). The stability and tat binding properties of these oligonucleotides were determined and compared, and the results are shown in Table 2 below.
  • Tm Melting temperature measurements were carried out in 100 mM NaCI/10 mM sodium phosphate buffer (pH 7.0). Samples were heated from 25 to 85 °C in 1 °C increments using a HP 8459 UV/VIS spectrophotometer and a HP 89100A temperature controller. The concentration of nucleic acid was 2.5-3.0 ⁇ M, and absorbance was monitored at 260 nm. T m values were determined by a first-derivative plot of absorbance vs temperature. Each experiment was performed in duplicate and the average reported as the thermal denaturation temperature. Ligand binding of the oligonucleotides was assessed by gel electrophoresis and RNA mobility shift assay.
  • Linker-derivatized oligoribonucleotides (5A-5CC) and the control sequences (4, 6 and 7, Fig.4) were 5'- 32 P-labeled with T4 polynucleotide kinase and [ - 32 P]ATP.
  • the labeled oligomers were then purified by phenol/chloroform extraction/EtOH precipitation or spin-column filtration (Bio-Rad, Bio-Spin 30). Prior to binding assays, the RNAs were dissolved in 20 mM Tris-HCl (pH 7.5)/ 100 mM NaCl, heated to 85 °C for 3 min, then slow-cooled to room temperature.
  • Binding assays were carried out in 20 ⁇ reaction mixtures containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5 U/ml RNAsin (Promega), 0.09 /_g/ml BSA, 5% (v/v) glycerol, 0.1 nM 32 P-labeled RNA (2000-5000 cpm) and either peptide derived from the HIV-1 Tat protein RKKRRQRRRPPQGS (amino acids 49- 62 of HIV LAI isolate) (Weeks et al., Science, 1990, 249:1281 ; Delling et al., Proc. Natl. Acad.
  • the gels were dried onto DEAE paper (Whatman DE81 ) and exposed to Kodak X-Omat X-ray film with an intensifying screen overnight at -70°C.
  • Competition binding experiments were carried out as described above except that the concentration of Tat protein was kept constant at 100 nM and unlabeled competitor RNA was added in a concentration range of 0.9 nM to 5000 nM.
  • K d values are expressed in nanomolar concentrations
  • Binding capacity indicates the % of active RNA molecules capable of binding to peptide upon saturation
  • linker- derivat ⁇ zed TAR analogue had some secondary structure. With the exception of structure 5C which incorporates a linker expected to be too short to allow proper duplex formation, binding assays revealed tat- binding function in the conjugated duplexes versus the unlinked controls. Similar binding was also confirmed in experiments using the full length tat protein. Further evaluation of linker incorporation has indicated that relatively short linkers can be used to advantage, to replace nucleotides resident in the polynucleotide strands, e.g. to replace nucleotides in the bulge of TAR.
  • Tat-binding analysis of the resulting structure has shown that replacement of nucleotides within the bulge preserved the tat-binding structure of TAR.
  • linkers equivalent in length to C 3 can be used, particularly within the so-called bulge structures which form at non-annealed sites of duplex structures.
  • the binding affinities of the Tar conjugates for full-length Tat protein were assessed using the mobility shift assay.
  • the K d value for the full- length Tat (1.17 nM) was slightly higher than that for the Tat-derived peptide (0.71 nM).
  • Tar conjugate 5B was added to a pre-formed complex between the 27mer fragment of the wild-type Tar stem-loop (oligomer 4) and full-length Tat protein, strong competition with the TAR sequence was observed. The complex was totally competed away when the ratio between the Tar conjugate and the Tat protein was 1 :1.
  • the linear conjugate 10 was first radiolabelled with gamma 32 P-ATP as described previously herein.
  • the heated T4 polynucleotide mixture was then cooled slowly to room temperature, and 1 ⁇ l (10 units) of T4 RNA ligase were then mixed with 10 /I of radiolabelled conjugate, 2 ⁇ of ATP (10mM) and 7 ul of 1X ligase buffer consisting of 66mM Tris-HCl (pH 7.5), 6.6 mM MgCI 2 , 1 mM DTT, and 1 mM ATP.
  • the ligation reaction was pursued for four hours at room temperature.
  • the ligated product was then purified on a 20% denaturing polyacrylamide gel.
  • the band corresponding to the cyclic conjugate (evident from its faster migration relative to linear conjugates) was cut out and extracted from the gel with 0.3 M NaOAc at room temperature overnight.
  • the sodium acetate solution containing the cyclic conjugate was then washed with an equal volume of phenol solution in order to eliminate any proteinaceous contamination. After this step, two volumes
  • a number of potential ligation sites were examined using structure 12 (Fig. 5).
  • the linear conjugates (one for each ligation site chosen) were synthesized and radiolabelled with gamma 32 P-ATP as described previously herein. 10 ⁇ of each radiolabelled conjugate was added to 2ul of ATP (10mM), 2 ⁇ of DMSO(100%), 2 ⁇ of 10X ligase buffer consisting of 500mM Tris-HCl (pH 7.8).
  • RNA Ligase 100n ⁇ M MgCI 2 lOOmM ⁇ -mercaptoethanol, 10mM ATP, and 1/.I (10 UNITS) of RNA Ligase.
  • the ligation reaction was pursued for 4 hours at 37°C.
  • the ligated products (2/ I of each) were examined by separation on 20% denaturing polyacrylamide and compared directly to an equivalent amount of unligated linear radiolabelled polynucleotide conjugate on the same gel.
  • Example 9- Binding properties of cyclic polynucleotide conjugates Using the best ligation site identified from the previous example, there was successfully generated a series of Tar conjugates; two of them are illustrated in Figure 6. Both of these constructs (14 & 15) are 21- mers and differ only In the chemical linker used to replace the nucleotide loops at the top and bottom of the duplex. Oligomer 14 contains Linker A and oligomer 15 contains linker D. All three cyclic polynucleotide conjugates were subjected to the binding assay as described previously.
  • the polynucleotides were purified on 20% denaturing polyacrylamide gels as previously described. For each of the various conditions, the same amount of radiolabelled gel-purified polynucleotide was used (300,000 CPM). The conditions used for each of the reactions are described below.
  • Exounuclease III 300,000 CPM of gel-purified polynucleotide was incubated in the presence of 20 units of Exonuclease III (1//I) and 1 /I of 10X buffer which consisted of 500mM Tris-HCl pH 8.0, 50mM MgCI 2 ,
  • Muno Bean Nuclease 300,000 CPM of gel-purified polynucleotide was incubated in the presence of 5 units of Mung Bean Nuclease (1/ I) and 1//I of 10X buffer which consisted of 500mM sodium acetate pH 5,0, 300mM NaCl, 1 mM ZnS0 4 . Enzymatic treatment was pursued for 6 h at 37°C and a sample was removed for analysis at this time.
  • Calf Intestinal Alkaline Phosphatase 300,000 of CPM gel-purified polynucleotide was incubated in the presence of 5 units of calf intestinal alkaline phoshatase and 1 l of 10X buffer which consisted of 500mM Tris-HCl pH 8.5, and 1 mM EDTA. Enzymatic treatment was pursued for 20 h at 37°C and a sample was removed at this time.
  • Cell and nuclear extracts were prepared essentially by the method of Dignam et. al., 1983, Nucl. Acids. Res., 11 :1475. The amount of protein in each extract was determined using Bovine Serum Albumin as a standard. 300,000 CPM of gel-purified polynucleotide was incubated in the presence of 8ug cell extract protein, or 6//g nuclear extract protein at 37 °C. Equivalent samples from both cell and nuclear extract digestions were removed at various times (8 and 24 h).
  • Tar conjugate 5A has a similar stability as the wild-type sequence (oligomer 4) in cellular and nuclear extracts although the conjugate appears far more stable against single strand-specific nucieases such as mung bean nucleases.
  • the duplex- forming cyclic linker molecules (oligomer 14 & 15) are much more stable than both the linear conjugates and the single-stranded cyclic control (oligomer 16).

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Abstract

Structures en double hélice à liaison de ligands présentant une stabilité sensiblement améliorée dans des conditions physiologiques. Les structures ont la forme de conjugués polynucléotidiques aptes à adopter une structure en double hélice dans laquelle des brins polynucléotidiques cyclisables sont couplés de manière covalente au niveau de l'une de leurs extrémités, ou des deux, par l'intermédiaire d'un linker chimique créant un pont stabilisateur entre les brins. On décrit diverses applications des structures stabilisées en double hélice, notamment des applications thérapeutiques, par exemple, dans le traitement du SIDA.
PCT/CA1992/000423 1991-09-27 1992-09-25 Conjugues polynucleotidiques a formation de doubles helices WO1993006122A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2273932A (en) * 1992-11-24 1994-07-06 Stiefel Laboratories Stable oligonucleotides
EP0804443A1 (fr) * 1995-01-18 1997-11-05 Pharmagenics, Inc. Oligomeres d'ester de phosphore non nucleotidiques
EP1219708A1 (fr) * 1999-10-08 2002-07-03 National Institute of Advanced Industrial Science and Technology Aptamere module et procede de detection d'une proteine cible a l'aide de celui-ci
US6504019B2 (en) 2000-03-24 2003-01-07 Bayer Corporation Nucleic acid probes having highly hydrophilic non-nucleosidic tags with multiple labels, and uses thereof
US6830890B2 (en) * 1994-10-24 2004-12-14 Affymetrix, Inc. Nucleic acid probe libraries
EP1724348A2 (fr) 1994-10-13 2006-11-22 Solexa, Inc. Supports en phase solide avec un tag d'ANP et d'amidate
JP2011050381A (ja) * 2009-08-07 2011-03-17 Gene Design Inc 新規オリゴヌクレオチド誘導体及びそれから成るＮＦ−κＢデコイ
WO2022246196A1 (fr) * 2021-05-21 2022-11-24 Olix Us, Inc. Réactifs de phosphorylation chimique, préparation et leurs utilisations

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1985000621A1 (fr) * 1983-07-22 1985-02-14 Koester Hubert Procede de reticulation definie de fragments d'adn a double helice au moyen d'un adn de liaison ainsi que d'oligonucleotides servant d'adn de liaison
WO1987007300A2 (fr) * 1986-05-23 1987-12-03 Worcester Foundation For Experimental Biology Inhibition de l'htlv-iii par des oligonucleotides exogenes

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1985000621A1 (fr) * 1983-07-22 1985-02-14 Koester Hubert Procede de reticulation definie de fragments d'adn a double helice au moyen d'un adn de liaison ainsi que d'oligonucleotides servant d'adn de liaison
WO1987007300A2 (fr) * 1986-05-23 1987-12-03 Worcester Foundation For Experimental Biology Inhibition de l'htlv-iii par des oligonucleotides exogenes

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Title
Dialog Information Services, file 155, Medline, Dialog Accession No. 05283440, Medline Accession No.84207440, Lathe R et al: "Linker tailing: unphos- phorylated linker oligonucleotides for joining DNA termini", DNA 1984, 3 (2) p 173-82 *
Dialog Information Services, file 155, Medline, Dialog Accession No. 07241406, Medline Accession No.90148406, Cieplak P et al: "Conformations of duplex structures formed by oligodeoxynucleotides covalent-ly linked to the intercalator 2-methoxy-6-chloro-9- aminoacridine", J Biomol Struct Dyn, Oct 1987, 5 (2) p 361-82 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2273932A (en) * 1992-11-24 1994-07-06 Stiefel Laboratories Stable oligonucleotides
EP1724348A2 (fr) 1994-10-13 2006-11-22 Solexa, Inc. Supports en phase solide avec un tag d'ANP et d'amidate
US6830890B2 (en) * 1994-10-24 2004-12-14 Affymetrix, Inc. Nucleic acid probe libraries
EP0804443A1 (fr) * 1995-01-18 1997-11-05 Pharmagenics, Inc. Oligomeres d'ester de phosphore non nucleotidiques
EP0804443A4 (fr) * 1995-01-18 1998-04-29 Pharmagenics Inc Oligomeres d'ester de phosphore non nucleotidiques
EP1219708A1 (fr) * 1999-10-08 2002-07-03 National Institute of Advanced Industrial Science and Technology Aptamere module et procede de detection d'une proteine cible a l'aide de celui-ci
EP1219708A4 (fr) * 1999-10-08 2004-08-11 Nat Inst Of Advanced Ind Scien Aptamere module et procede de detection d'une proteine cible a l'aide de celui-ci
US6504019B2 (en) 2000-03-24 2003-01-07 Bayer Corporation Nucleic acid probes having highly hydrophilic non-nucleosidic tags with multiple labels, and uses thereof
JP2011050381A (ja) * 2009-08-07 2011-03-17 Gene Design Inc 新規オリゴヌクレオチド誘導体及びそれから成るＮＦ−κＢデコイ
WO2022246196A1 (fr) * 2021-05-21 2022-11-24 Olix Us, Inc. Réactifs de phosphorylation chimique, préparation et leurs utilisations

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