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WO1996011010A1 - Oligonucleotides a double helice ou a quadruplex avec ou sans liaison - Google Patents

Oligonucleotides a double helice ou a quadruplex avec ou sans liaison Download PDF

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Publication number
WO1996011010A1
WO1996011010A1 PCT/US1995/011985 US9511985W WO9611010A1 WO 1996011010 A1 WO1996011010 A1 WO 1996011010A1 US 9511985 W US9511985 W US 9511985W WO 9611010 A1 WO9611010 A1 WO 9611010A1
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Prior art keywords
oligodeoxyribonucleotide
seq
duplex
quadruplex
thrombin
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PCT/US1995/011985
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English (en)
Inventor
Roman F. Macaya
Hetian Gao
Michael E. Joesten
Bruce A. Beutel
Arthur H. Bertelsen
Alan F. Cook
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Pharmagenics, Inc.
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Application filed by Pharmagenics, Inc. filed Critical Pharmagenics, Inc.
Priority to AU36369/95A priority Critical patent/AU3636995A/en
Publication of WO1996011010A1 publication Critical patent/WO1996011010A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • C12N2310/151—Nucleic acids forming more than 2 strands, e.g. TFOs more than 3 strands, e.g. tetrads, H-DNA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the serine protease thrombin plays a critical role
  • DNA oligonucleotides conform to a consensus sequence which folds into a unimolecular DNA quadruplex containing two G quartets held together by three loops (Macaya et al . , 1993,- Schultze et al . , 1994; and Wang et al . , 1993).
  • quadruplex/duplex oligonucleotides were tested for thrombin binding and inhibitory activity. In comparative testing, all quadruplex/duplex molecules bound to thrombin with higher affinity than molecules containing only the
  • Figure 1A DNA sequences that bind with high affinity to thrombin.
  • Bold letters identify the sequences which conform to the previously identified consensus sequence (Bock et al . , 1992) and underlined bases identify the complementary sequences on either side of the quadruplex consensus.
  • Figure 1B Consensus structure of a quadruplex I5mer which has been previously identified from a selection for DNA ligands to thrombin (Bock et al . , 1992). Rectangles indicate the G residues involved in quadruplex formation. Shaded rectangles indicate syn G nucleotides. N indicates a variable base composition. The dash between the two T's indicates a T-T base pair.
  • Figure 1C One possible folded conformation of a consensus sequence that binds tightly to thrombin.
  • N indicates a variable base composition and N' indicates its Watson-Crick base pairing partner.
  • Figure 1D Dimeric quadruplex/duplex structure formed by unbridged complementary base pairing of the sequences flanking the quadruplex motif.
  • Figures 1E and 1F Structures of quadruplex/duplex motifs examined (See Table 1 for details) .
  • Figure 2A Binding curves for DNA oligonucleotides to thrombin. Sequences and structures can be identified from Table 1. Plot of percent DNA bound as a function of thrombin concentration. Symbols for oligonucleotides are as follows: ( ⁇ ) 15mer quadruplex (SEQ ID NO: 21); ( ⁇ ) 24mer quadruplex/duplex (SEQ ID NO: 22)); ( ⁇ ) 24mer with triethylene glycol (TEG) bridge, nicked stem (SEQ ID NO: 23); ( ⁇ ) 24mer, TEG bridge, nicked quadruplex (SEQ ID NO: 24); ( ⁇ ) 32mer, TEG bridge, nicked 8 base-pair stem (SEQ ID NO: 26) ( ⁇ ) 32mer, 8 base-pair stem (SEQ ID NO: 27); ( ⁇ 48mer unimolecular "dimer mimetic", 2 TEG bridges, 1 nicked 4 base-pair stem (SEQ ID NO: 31); (v) 48mer "d
  • Figure 2B Binding competition studies of 32 P-labeled 15mer (SEQ ID NO: 21) vs DNA oligonucleotides. Competing
  • unlabeled oligonucleotides used were: ( ⁇ ) 15mer (SEQ ID NO: 21); ( ⁇ ) quadruplex/duplex with TEG-bridged, ligated 8 base-pair stem (SEQ ID NO: 28); ( ⁇ ) unimolecular "dimer mimetic" (SEQ ID NO: 30); ( ⁇ ) scrambled 15mer d-GGTGGTGGTTGTGGT (SEQ ID NO: 19). Percentage binding was calculated for labeled 15mer as described in Materials and Methods. These determinations were done in duplicate and error bars are shown.
  • Figure 3A-3C One dimensional ⁇ NMR spectra of the H- bonded imino and amino proton resonances of (A,B) 24mer quadruplex/duplex d(CTACTGGTTGGTGAGGTTGGGTAG) with a 4 base-pair stem (SEQ ID NO: 22; see Table 1) or (C) 32mer quadruplex/duplex (SEQ ID NO: 28), acquired at 500 MHz as the first increment of an SS NOESY (Smallcombe, 1993) in 90% H 2 O/10% D 2 O at 1°C.
  • H-bonded imino proton resonances were identified as Watson-Crick T iminos (T), Watson-Crick G iminos (G) , Hoogsteen base paired quadruplex G iminos (Q), or T-T base-pair T iminos (T-T).
  • Sample conditions were: (A) 1.9 mM DNA, 88 mM KCl, (B) 0.12 mM DNA, 6.9 mM MgCl 2 , 6.1 mM KCl, (C) 1.3 mM DNA, 100 mM KCl . All samples were at pH 6.1. Sweep widths were: (A,B) 11,000 Hz or (C)
  • Figure 3E Region of imino-imino NOE crosspeaks from an SS NOESY spectrum of the same sample as (C). Sequential imino-imino NOE connectivities along the duplex stem are indicated by lines above the diagonal. The NOE crosspeaks between Hoogsteen base paired G iminos from adjacent quartets and the NOE crosspeaks between the T iminos from the T-T base pair and the sequential G iminos in the adjacent quartet are indicated below the diagonal.
  • the SS NOESY was acquired with 2048 complex points, a relaxation delay of 1.9 sec, a mixing time of 100 msec, a sweep width of 10,000 Hz, 400 t, increments and 128 scans per t 1 value. All the above spectra were collected using a 208 ⁇ sec SS pulse (Smallcombe, 1993), and processed by linear predicting the first 2 data points of the FID, solvent subtracting the residual water peak, and apodized using a gaussian window function.
  • Figure 4 Plot of percent oligonucleotide remaining vs. incubation time (hours) in human serum pooled from three individuals. Oligonucleotide sequences are identified from their corresponding numbers in Table 1.
  • the invention provides for single-stranded DNA oligonucleotide ligands to the serine protease thrombin. All quadruplex/duplex molecules tested bound to thrombin with higher affinity than quadruplex structures lacking the duplex-stem. However, binding affinities did not always correlate to inhibitory potency as some molecules with high affinity were not potent inhibitors in vitro. 1H NMR spectroscopy studies demonstrated that the duplex stem on these structures allows them to form
  • quadruplex/duplex structures by bridging the end of the duplex-stem improved their thrombin inhibitory activity. All constrained quadruplex/duplex molecules which were tested were more potent inhibitors than the quadruplex motif alone. In addition, bridging these structures
  • the present invention provides single stranded oligodeoxyribonucleotides that bind to thrombin upon contact and have a quadruplex consensus motif flanked by complementary sequences that form a duplex-stem motif.
  • a bridging group is bound to the end of the duplex-stem.
  • the duplex-stem preferably has at least four complementary base pairs, particularly about 4 to 20. These complementary base pair sequences are selected from, but not limited to, the group consisting of SEQ ID NOS: 1-18, 22-29 and 33-43, particularly oligonucleotides
  • the invention provides single stranded oligodeoxyribonucleotides that bind to thrombin upon contact and comprise a quadruplex consensus motif whose 5' and 3' termini are connected by a bridging moiety.
  • These oligodeoxyribonucleotides are exemplified by SEQ ID NO: 32.
  • the invention provides single stranded oligodeoxyribonucleotides that bind to thrombin upon contact and comprise two quadruplex motifs connected by duplex-stem motifs as shown in Figure IF, where one or both of these duplex-stem motifs are bridged by a non-nucleotide bridging group such as a glycol or disulfide group.
  • These oligodeoxyribonucleotides are preferably selected from, but are not limited to, SEQ ID NOS: 30 and 31.
  • Another aspect provides a method of inhibiting
  • thrombin activity by contacting thrombin with an inhibitory amount of an oligodeoxyribonucleotide of the invention preferably selected from the group consisting of SEQ ID NOS: 1-18, 22-29 and 30-43, and particularly selected from groups consisting of SEQ ID NOS: 26-28, 30-32 or 41-43.
  • the method can be performed in an individual in need thereof by administering to the individual a thrombin activity inhibitory amount of a oligodeoxyribonucleotide of the invention.
  • the oligonucleotide preferably comprises at least one sequence selected from the group consisting of SEQ ID NOS: 1-18, 22-29, 30-32 and 33-43, particularly one or more of SEQ ID NOS. 26-28, 30-32 or 41-43.
  • oligonucleotides of the present invention may be synthesized by a variety of accepted means known to those skilled in the art.
  • the oligonucleotides may be synthesized on an automated nucleic acid synthesizer.
  • the oligonucleotides may be synthesized enzymatically through the use of flanking or primer
  • oligonucleotides may be synthesized by solution phase chemistry. It is to be understood, however, that the scope of the present invention is not to be limited to any particular means of synthesis.
  • oligonucleotides of the present invention may be administered in conjunction with an acceptable
  • Such pharmaceutical compositions may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Such oligonucleotides may be
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form.
  • aqueous solutions of the active compounds in water-soluble or water-dispersible form.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the compounds of the present invention may also be administered encapsulated in liposomes, wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the active ingredient depending upon its solubility, may be present both in the aqueous layer, in the lipidic layer, or in what is generally termed a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises
  • phospholipids such as lecithin and sphingomycelin, steroids such as cholesterol, surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • the diameters of the liposomes generally range from about 15 nm to about 5 microns.
  • the oligonucleotides of the present invention may also be employed as diagnostic probes for determining the presence of thrombin, and thereby determining the need for modulation of its function or activity. In such
  • a modified or unmodified oligonucleotide of the present invention is added to a sample suspected of containing the thrombin.
  • the oligonucleotide may be labeled with a detectable marker such as a radioactive label, a chromogen, or an enzyme label.
  • oligonucleotide may be employed in a variety of assay methods for the detection of thrombin, such methods
  • oligonucleotide or an expression vector containing a binding sequence may be administered to a cell in order to prevent the deleterious consequences of overproduction or to effect the benefits of inhibiting the thrombin function.
  • the oligonucleotide may be administered to an individual to prevent clotting or extend clotting time.
  • Administration can be by any of a number of procedures known to those skilled in the art, including, but not limited to, transfection, electroporation, lipofection, transformation, or transduction with eukaryotic expression vectors such as viruses, including retroviruses, Herpes viruses, adenoviruses, and adeno-associated viruses.
  • eukaryotic expression vectors such as viruses, including retroviruses, Herpes viruses, adenoviruses, and adeno-associated viruses.
  • Heterologous or autologous transplants of such cells may be administered to patients by procedures knowtH to those skilled in the art.
  • telomere length has been implicated in the regulation of cell division, as cells which do not show telomerase activity, and therefore do not maintain telomere length, cannot divide indefinitely, whereas cells immortalized in vitro do show telomerase activity (Counter et al . , 1994).
  • Quadruplex DNA structures have been shown to inhibit telomerase activity in vitro (Zahler et al., (1991). Such quadruplex telomerase
  • inhibitors may prove to be novel antitumor drugs by
  • dimethoxytrityl triethylene glycol phosphoramidite were purchased from Glen Research, Sterling, VA.
  • Oligonucleotide HPLC purifications were performed using a Waters 600E system controller equipped with a multi-solvent delivery system and a model 991 photodiode array detector. Trityl-on separations were performed using a Waters RCM (8 mm ⁇ 10 cm) C 4 column for analytical purposes and a C 4 RCM
  • Oligonucleotides comprising consensus sequences were prepared on an Applied Biosystems model 394 DNA synthesizer on a 1 ⁇ mol scale. Unmodified oligonucleotides were purified by trityl-on HPLC followed by detritylation, extraction, ethanol precipitation and conversion to the potassium salt using AG50W-X8 resin. Triethylene-glycol-bridged oligonucleotides were prepared as previously described (Gao et al . , 1994). Disulfide-bridged
  • oligonucleotides were prepared as follows. For each sequence, four 1 ⁇ mol scale syntheses were carried out on a DNA synthesizer. For introduction of the masked thiol at the 3' -terminus, the thiol modifier-C 6 S-S (0.1 M in acetonitrile) was directly coupled to a standard solid support, with the coupling time being extended to 15 min in this coupling cycle and the following two nucleoside phosphoramidite coupling cycles. Conventional nucleoside phosphoramidites were then incorporated at the appropriate positions and the thiol modifier was added to the 5'-end of the oligonucleotide using a 15 min coupling time. Coupling efficiencies of about 98% were obtained for standard nucleoside phosphoramidites, and about 90% for the thiol modifier-C 6 , as determined by trityl assay. After
  • the crude oligonucleotide mixture in concentrated aqueous ammonia solution was left at 55° overnight to remove the base protecting groups. After removal of ammonia, the solutions were combined, concentrated to a small volume and the tritylated material was purified by HPLC on a preparative C 4 column using a linear gradient of 2-40% B over 40 min. The product peak was concentrated to a small volume, redissolved in potassium phosphate buffer (0.1 M, pH 8.0) and after addition of DTT (1 mg/OD 260) ), the mixture was stirred at room temperature for 4 hr to cleave the
  • the disulfhydryl oligonucleotide in 0.1 M sodium phosphate (pH 8) containing 0.3 M NaCl was oxidizecf in air at 4° for three days either at 4° or room temp. Analysis of the reaction mixture by PAGE showed that three bands were present. The required material was isolated by loading the mixture onto one half of a preparative gel (21 ⁇ 12 cm, 1.5 mm thick). The lower band corresponding to the disulfide-bridged oligonucleotide was cut out, frozen at -70°, crushed in 10 mM Tris (pH 7.5) and extracted with 0.3 M sodium acetate solution (pH 5.2). The
  • oligonucleotide was isolated from the gel slurry by spin filtration using a 0.45 ⁇ pore size cellulose acetate sterilization spin filtration unit followed by
  • DNA oligonucleotides to be tested were labeled with 32 P using T4 polynucleotide kinase and 32 P- ⁇ -ATP and purified on an 8% denaturing PAGE gel. Labeled DNA oligonucleotides were cut out from the gels and counted in a liquid
  • Stock thrombin (4,080 units/mg protein, 34 mM from Sigma) was diluted to 680, 68 and 6.8 nM in 20 mL of selection buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM tris acetate pH 7.4) containing
  • oligonucleotides usually about 30,000 cpm. Mixtures were incubated for 30 min at room temperature and then filtered through nitrocellulose filters on 12 -well filter manifolds. Labeled DNA retained on filters was counted and the percent DNA bound was determined using inputs onto filters as 100% bound and subtracting out background binding on filters (cpm retained in absence of protein) .
  • d(GGTTGGTGTGGTTGG) was labeled and purified as described above.
  • Competition binding studies were carried out by determining the percent 15mer DNA (SEQ ID NO: 21) bound at 680 nM thrombin as described above, in the presence of 0, 5, 10, 20, 40, or 100 picomoles of unlabeled competitor DNA. 84 fmoles of labeled 15mer were used as input.
  • thrombin-catalyzed fibrin clot formation was performed as previously described (Bock et al . , 1992).
  • Thrombin enzyme with an activity of 4,080 units/mg protein and fibrinogen (fraction 1, type 1 from human plasma) were purchased from Sigma.
  • 200 ⁇ L of human fibrinogen in selection buffer 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM tris acetate pH 7.4
  • selection buffer 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM tris acetate pH 7.4
  • the reaction was initiated with the addition of 100 ⁇ L of human thrombin (also equilibrated in selection buffer to 37°C for one minute) for a final concentration of 2 mg/mL fibrinogen and 13 nM thrombin in 300 ⁇ L. Time was stopped at the first indication of a clot. Clotting was determined by observing the appearance of a small clot on a hooked wire which was manually dipped into the reaction mixture
  • Clot formation was usually complete within 5 seconds of the first appearance of a clot.
  • the elution gradient was 17% acetonitrile over 15 min, 17-40% over 10 min and held at 40% for 5 min, then 40-70% over 1 min and held at 70% for 3 min.
  • the retention times for fibrinopeptides A and B were determined from pure standards purchased from Sigma. Since only fibrinopeptide A eluted as a well resolved peak, only the release of this peptide was studied quantitatively by the integration of its UV absorption peak using Millenium 2010 Chromatography Manager V2.00 system software.
  • 1H NMR spectra were acquired at 500 MHz on a Varian UNITY plus spectrometer.
  • One dimensional spectra in 90% H 2 O/10% D 2 O were acquired using symmetrically-shifted (SS) shaped pulses to suppress the water resonance (Smallcombe 1993).
  • P. COSY spectra were acquired using a full 90° flip angle pulse (Marion & Bax, 1988).
  • NOESY spectra in D 2 O were acquired in the phase-sensitive mode (States et al . 1982) using the standard pulse sequence (Kumar et al. 1980) and with presaturation of the water resonance during the recycle delay.
  • SS NOESY spectra in water were acquired in the phase-sensitive mode using symmetrically-shifted shaped pulses (Smallcombe 1993). Spectra were processed using Varian VNMR processing software version 4.2A. Linear prediction was used eliminate large frequency-dependent phase shifts from spectra acquired using SS pulses.
  • Figure 1E illustrates the monomer and Figure 1F the "dimer mimetic".
  • L1, L2 indicate the base sequences in the corresponding loops of the quadruplex.
  • X indicates the base sequences attached to the 5' end (the left side) of the quadruplex-duplex junction and SEQ ID NO: 40 further has a TGC sequence at the quadruplex-duplex junction Y.
  • B1 indicates the type of bridge, a single entry indicates that only one bridge (B1) is present [triethylene glycol (TEG) or disulfide (-S-S-)].
  • SEQ ID NO: 31 further has a TEG bridge at B2.
  • "Nick" indicates the position in the diagram of any unligated 5'- and 3' ends.
  • a "nick” in duplex DNA is the absence of a phosphodiester bond between two adjacent nucleotides of one strand. Sequences with SEQ ID NOS: 23-26, 28, 30 and 31 contain a triethylene glycol bridge at the end of the duplex structure. "#bp” indicates the number of base pairs in the stem (if zero, there is no T nucleotide between the quadruplex and duplex motifs). "Stem Sequence” refers to the sequence in the stem starting at the N1 position in Figure 1E. "N'" is the complementary base.
  • Oligonucleotides containing both the quadruplex and duplex-stem motifs (SEQ ID NOS: 22, 23, 27 and 28) bound to thrombin with nearly two fold higher affinity than the isolated quadruplex motif (SEQ ID NO: 21) (Fig. 2A).
  • SEQ ID NO: 21 isolated quadruplex motif
  • quadruplex motif (SEQ ID NO: 24) bound poorly, probably due to the inability of such a "nicked" quadruplex to form a stable structure (Fig. 2A). This result indicates that although the duplex-stem motif can enhance binding in quadruplex/duplex molecules, it is not sufficient by itself for thrombin binding.
  • a competition study (Fig. 2B) determined that the higher affinity exhibited by
  • oligonucleotides is not due to non-specific binding.
  • the higher affinity of the quadruplex/duplex structure is therefore probably due to additional or optimal protein contacts at the binding site on thrombin.
  • DNA and thrombin concentrations are shown as nanomolar amounts.
  • the unimolecular quadruplex/duplex structure could be considered a hairpin with a highly structured loop, thus making the quadruplex motif the most structured loop reported to date.
  • the formation of the multimeric structure described above may be limited to molecules which are not kinetically trapped as a stable unimolecular structure.
  • Oligonucleotides which were capable of folding into unimolecular "dimer mimetics" were prepared in order to determine the binding and inhibitory activities of these types of structures. These dimer mimetics consisted of two 24mer oligonucleotides (of the same sequence as SEQ ID NO: 22) in tandem, with a tri-ethylene glycol linker spanning one (SEQ ID NO: 30) or both (SEQ ID NO: 31) duplex-stem(s)
  • a series of circular (no nick) oligonucleotides was synthesized in which the 5' and 3' ends to the duplex-stem were covalently linked by either a triethylene glycol (TEG) bridge (SEQ ID NOS: 25 and 28) or a disulfide-containing polymethylene bridge (-S-S-) (SEQ ID NOS: 33, 34 and 39-43).
  • TAG triethylene glycol
  • -S-S- disulfide-containing polymethylene bridge
  • oligonucleotides with sulfhydryl groups attached to the ends via aliphatic, six carbon linkers were prepared and the disulfide bridges were produced by oxidation in air.
  • This procedure was effective in producing bridges in a wide variety of oligonucleotides (SEQ ID NOS: 33, 34 and 36-43) even when the base paired region was very short. In fact a bridged quadruplex without any stem (SEQ ID NO: 32) could be prepared in good yield by this procedure.
  • quadruplex/duplex molecule (SEQ ID NO: 28) was at least as effective at inhibiting thrombin as a five-fold higher concentration of the isolated quadruplex (SEQ ID NO: 20) was also determined using a fibrinopeptide release assay (Table 4) .
  • Thrombin concentration was 6 nM. "% Fib. A Release” indicates the percent of fibrinopeptide A released as compared to control (no oligo) reaction.
  • SEQ ID NO: 28 inhibited the thrombin-catalyzed release of fibrinopeptide A from fibrinogen slightly more effectively than SEQ ID NO: 20 at 100 nM (Table 4).
  • the activity of thrombin at 6 nM was inhibited by 50% (IC 50 ) with the quadruplex/duplex molecule SEQ ID NO: 28 (bridged, 8 base-pair stem) at a concentration of 6 nM in the clotting assay described above (data not shown).
  • the relevance of the stem sequence and length to the inhibitory activity of these structures was investigated by synthesizing a series of disulfide-bridged quadruplex/duplex molecules with 0, 2, 4, 5, 6, 7, 8, 9, and 10 base-pair stems (SEQ ID NOS: 32-34, 36, 37 and 41-43 respectively), and oligonucleotides with three different sequences of the 7 base-pair stem (SEQ ID NOS: 38-40).
  • the inhibitory activity increased with length up to 4 base-pairs, with a slight reduction at 5 and 6 base-pairs, and then an increase through 10 base-pairs (Table 3).
  • the inhibitory activity varied among three quadruplex/duplex molecules containing different 7 base-pair stem sequences (SEQ ID NOS: 38-40) and two
  • duplex-stem motif is, therefore, a second structural entity which can be optimized for thrombin inhibitory activity. Without this second motif, optimization of thrombin ligands is limited to modifications of the quadruplex motif alone.
  • Oligonucleotides with relatively high activities in the clotting assay were incubated in human serum and aliquots were analyzed using an ion exchange HPLC method to determine the extent of degradation. Using this procedure, the unbridged 15mer SEQ ID NO: 20 was shown to be rapidly degraded with a half-life of approximately 17 minutes. This relatively short half-life is consistent with data reported for other natural DNA oligonucleotides (Akhtar et al.,
  • sequences with superior activity in the inhibition of clot formation also possess substantially longer half-lives in serum suggests that they may be candidates for therapeutic use.
  • Oligonucleotides are identified by their corresponding SEQ ID NO: Table 1.

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Abstract

Oligodésoxyribonucléotide monocaténaire se fixant à la thrombine par contact et comprenant un motif structural consensus à quadruplex flanqué par des séquences complémentaires constituant un motif structural à tige en double hélice. Oligodésoxyribonucléotide se fixant à la thrombine par contact et comprenant un motif structural consensus à quadruplex, dont les terminaisons 5' et 3' sont reliées par une fraction de liaison. Procédés d'inhibition et de détection de la thrombine au moyen d'un ou de plusieurs de ces oligodésoxyribonucléotides.
PCT/US1995/011985 1994-10-07 1995-09-20 Oligonucleotides a double helice ou a quadruplex avec ou sans liaison WO1996011010A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997020924A1 (fr) * 1995-12-04 1997-06-12 Saicom S.R.L Classe d'oligonucleotides, utiles du point de vue therapeutique comme agents anti-tumoraux
WO1997048715A1 (fr) * 1996-06-19 1997-12-24 Hybridon, Inc. Modulation de la formation de tetraplex par modifications chimiques d'un oligonucleotide contenant g¿4?
WO1998004571A3 (fr) * 1996-07-26 1998-04-16 Hybridon Inc Purification par affinite d'oligonucleotides au moyen d'oligonucleotides multimeres solubles
WO2003002592A1 (fr) * 2001-06-29 2003-01-09 Unisearch Limited Aptameres et antiaptameres
EP1434882A4 (fr) * 2001-09-24 2006-02-22 Ingeneus Corp Aptameres contenant des sequences d'acides nucleiques ou d'analogues d'acides nucleiques liees de maniere homogene ou dans des nouveaux complexes
WO2014098773A1 (fr) * 2012-12-21 2014-06-26 Nanyang Technological University Induction site-spécifique d'hybrides bimoléculaires quadruple brin-double brin et procédés d'utilisation de ceux-ci
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs

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* Cited by examiner, † Cited by third party
Title
BRITISH JOURNAL OF CANCER, Volume 60, issued 1989, TIDD et al., "Partial Protection of Oncogene, Anti-sense Oligodeoxynucleotides Against Serum Nuclease Degradation Using Terminal Methylphosphonate Groups", pages 343-350. *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Volume 114, issued 1992, GLICK et al., "Trapping and Isolation of an Alternate DNA Conformation", pages 5447-5448. *
LETTERS TO NATURE, Volume 355, issued 06 February 1992, BOCK et al., "Selection of Single-stranded DNA Molecules that Bind and Inhibit Human Thrombin", pages 564-566. *
NUCLEIC ACIDS RESEARCH, Volume 18, No. 21, issued 1990, DURAND et al., "Circular Dichroism Studies of an Oligodeoxyribonucleotide Containing a Hairpin Loop Made of a Hexaethylene Glycol Chain: Conformation and Stability", pages 6353-6359. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 90, issued April 1993, MACAYA et al., "Thrombin-binding DNA Aptamer Forms a Unimolecular Quadruplex Structure in Solution", pages 3745-3749. *
THE EMBO JOURNAL, Volume 8, No. 13, issued 1989, CASE et al., "The Unusual Stability of the IS10 Anti-sense RNA is Critical for Its Function and is Determined by the Structure of Its Stem-domain", pages 4297-4305. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, No. 24, issued 25 August 1993, PADMANABHAN et al., "The Structure of Alpha-Thrombin Inhibited by a 15-Mer Single-stranded DNA Aptamer", pages 17651-17654. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997020924A1 (fr) * 1995-12-04 1997-06-12 Saicom S.R.L Classe d'oligonucleotides, utiles du point de vue therapeutique comme agents anti-tumoraux
WO1997048715A1 (fr) * 1996-06-19 1997-12-24 Hybridon, Inc. Modulation de la formation de tetraplex par modifications chimiques d'un oligonucleotide contenant g¿4?
WO1998004571A3 (fr) * 1996-07-26 1998-04-16 Hybridon Inc Purification par affinite d'oligonucleotides au moyen d'oligonucleotides multimeres solubles
WO2003002592A1 (fr) * 2001-06-29 2003-01-09 Unisearch Limited Aptameres et antiaptameres
EP1434882A4 (fr) * 2001-09-24 2006-02-22 Ingeneus Corp Aptameres contenant des sequences d'acides nucleiques ou d'analogues d'acides nucleiques liees de maniere homogene ou dans des nouveaux complexes
WO2014098773A1 (fr) * 2012-12-21 2014-06-26 Nanyang Technological University Induction site-spécifique d'hybrides bimoléculaires quadruple brin-double brin et procédés d'utilisation de ceux-ci
US10822607B2 (en) 2012-12-21 2020-11-03 Nanyang Technological University Site-specific induction of bimolecular quadruplex-duplex hybrids and methods of using the same
US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
US12269839B2 (en) 2017-06-30 2025-04-08 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use

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