EP0579771A1 - Oligonucleotides circulaires a brin unique - Google Patents

Oligonucleotides circulaires a brin unique

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Publication number
EP0579771A1
EP0579771A1 EP92912127A EP92912127A EP0579771A1 EP 0579771 A1 EP0579771 A1 EP 0579771A1 EP 92912127 A EP92912127 A EP 92912127A EP 92912127 A EP92912127 A EP 92912127A EP 0579771 A1 EP0579771 A1 EP 0579771A1
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EP
European Patent Office
Prior art keywords
target
oligonucleotide
circular
domain
analog
Prior art date
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EP92912127A
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German (de)
English (en)
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EP0579771A4 (en
Inventor
Eric T. Kool
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Research Corp Technologies Inc
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Research Corp Technologies Inc
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Publication of EP0579771A1 publication Critical patent/EP0579771A1/fr
Publication of EP0579771A4 publication Critical patent/EP0579771A4/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
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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
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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
    • C12N15/1135—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 oncogenes or tumor suppressor genes
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • 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
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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
    • C12N2310/152—Nucleic acids forming more than 2 strands, e.g. TFOs on a single-stranded target, e.g. fold-back TFOs
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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
    • C12N2310/3183—Diol linkers, e.g. glycols or propanediols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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 present invention provides single-stranded circular oligonucleotides capable of binding to a target DNA or RNA and thereby regulating DNA replication, RNA transcription, protein translation, and other processes involving nucleic acid templates. Furthermore, circular oligonucleotides can be labeled for use as probes to detect or isolate a target nucleic acid. Circular oligonucleotides can also displace one strand of a duplex nucleic acid without prior denaturation of the duplex. Moreover, circular oligonucleotides are resistant to exonucleases and bind to a target with higher selectivity and affinity than do linear
  • An oligonucleotide binds to a target nucleic acid by forming hydrogen bonds between bases in the target and the oligonucleotide.
  • Common B DNA has conventional adenine-thymine (A-T) and guanine-cytosine (G-C) Watson and Crick base pairs with two and three hydrogen bonds, respectively.
  • A-T adenine-thymine
  • G-C guanine-cytosine
  • hybridization technology is based upon the capability of sequence-specific DNA or RNA probes to bind to a target nucleic acid via Watson-Crick hydrogen bonds.
  • other types of hydrogen bonding patterns are known wherein some atoms of a base which are not involved in Watson-Crick base pairing can form hydrogen bonds to another nucleotide.
  • thymine (T) can bind to an A-T Watson-Crick base pair via hydrogen bonds to the adenine, thereby forming a T-AT base triad.
  • oligonucleotide with the necessary structural features to achieve stable target binding with both Watson-Crick and alternate hydrogen bonds.
  • Oligonucleotides 'have been observed to bind by non-Watson-Crick hydrogen bonding in vitro.
  • Cooney et al., 1988, Science 241: 456 disclose a 27-base single-stranded oligonucleotide which bound to a double-stranded nucleic acid via non-Watson-Crick hydrogen bonds.
  • triple-stranded complexes of this type are not very stable, because the oligonucleotide is bound to its target only with less stable alternate hydrogen bonds, i.e., without any
  • Oligonucleotides have been used for a variety of utilities. For example, oligonucleotides can be used as probes for target nucleic acids that are immobilized onto a filter or membrane, or are present in tissues. Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY) provide a detailed review of hybridization techniques.
  • oligonucleotides As regulators of cellular nucleic acid biological function. This interest arises from observations on naturally occurring complementary, or antisense, RNA used by some cells to control protein expression.
  • RNA complementary, or antisense, RNA used by some cells to control protein expression.
  • the development of oligonucleotides for in vivo regulation of biological processes has been hampered by several long-standing problems, including the low binding stability and nuclease sensitivity of linear oligonucleotides.
  • transcription of the human c-myc gene has been inhibited in a cell free, in vitro assay system by a 27-base linear oligonucleotide designed to bind to the c-myc promoter. Inhibition was only
  • linear oligonucleotides were used to inhibit human immunodeficiency virus replication in cultured cells.
  • Linear oligonucleotides complementary to sites within or near the terminal repeats of the retrovirus genome and within sites complementary to certain splice junctions were most effective in blocking viral replication.
  • these experiments required large amounts of the linear oligonucleotides before an effect was obtained,
  • oligonucleotides that are useful as regulators of biological processes preferably possess certain properties.
  • the oligonucleotide should bind strongly enough to its complementary target nucleic acid to have the desired regulatory effect.
  • the oligonucleotide and its target be sequence specific.
  • the oligonucleotide should have a sufficient half-life under in vivo conditions for it to be able to accomplish its desired regulatory action in the cell.
  • oligonucleotide should be resistant to enzymes that degrade nucleic acids, e.g. nucleases. Fourth, the oligonucleotide should be able to bind to single- and double-stranded targets .
  • linear oligonucleotides may satisfy the requirement for sequence specificity, linear
  • oligonucleotides are sensitive to nucleases and
  • linear oligonucleotides bind to form a two-stranded complex like those present in cellular nucleic acids. Consequently, cellular enzymes can readily manipulate and dissociate a linear oligonucleotide bound in a double-stranded complex with target.
  • oligonucleotides can bind to a double-stranded target via alternate hydrogen bonds (e.g. Hoogsteen binding), linear oligonucleotides cannot readily dissociate a double-stranded target to replace one strand and thereby form a more stable Watson-Crick bonding pattern.
  • an oligonucleotide with high binding affinity can be used at lower dosages.
  • the present invention provides single-stranded circular oligonucleotides which, by nature of the circularity of the oligonucleotide and the domains present on the oligonucleotide, are nuclease resistant and bind with strong affinity and high selectivity to their targeted nucleic acids. Moreover, the present circular oligonucleotides can dissociate and bind to a double-stranded target without prior
  • Single-stranded circles of DNA or RNA are known.
  • the structures of some naturally occurring viral and bacteriophage genomes are single-stranded circular nucleic acids.
  • Single-stranded circles of DNA have been studied by Erie et al. (1987, Biochemistry 26: 7150-7159 and 1989, Biochemistry 28: 268-273). However, none of these circular molecules are designed to bind a target nucleic acid.
  • the present invention represents an innovation characterized by a substantial improvement relative to the prior art since the subject circular oligonucleotides exhibit high specificity, low or no toxicity and more resistance to nucleases than linear oligonucleotides, while binding to single- or double-stranded target nucleic acids more strongly than conventional linear oligonucleotides.
  • the present invention provides a single-stranded circular oligonucleotide having at least one parallel binding (P) domain and at least one anti-parallel binding (AP) domain, and having a loop domain between each binding domain to form the circular
  • Each P and corresponding AP domain has sufficient complementarity to bind detectably to one strand of a defined nucleic acid target with the P domain binding in a parallel manner to the target, and the AP domain binding in an anti-parallel manner to the target.
  • Sufficient complementarity means that a sufficient number of base pairs exists between the target nucleic acid and the P and/or AP domains of the circular oligonucleotide to achieve stable, i.e.
  • Another aspect of the present invention provides the subject single-stranded circular
  • oligonucleotides derivatized with a reporter molecule to provide a probe for a target nucleic acid, or with a drug or other pharmaceutical agent to provide cell specific drug delivery, or with agents which can cleave or otherwise modify the target nucleic acid or,
  • An additional aspect of the present invention provides single-stranded circular oligonucleotides linked to a solid support for isolation of a nucleic acid complementary to the oligonucleotide.
  • Another aspect of the present invention provides a compartmentalized kit for detection or diagnosis of a target nucleic acid including at least one first container providing any one of the present circular oligonucleotides.
  • a further aspect of the present invention provides a method of detecting a target nucleic acid which involves contacting a single-stranded circular oligonucleotide with a sample containing the target nucleic acid, for a time and under conditions sufficient to form an oligonucleotide-target complex, and detecting the complex.
  • This detection method can be by
  • a still further aspect of the present invention provides a method of regulating biosynthesis of a DNA, an RNA or a protein. This method includes contacting at least one of the subject circular
  • oligonucleotides with a nucleic acid template for the DNA, the RNA or the protein under conditions sufficient to permit binding of the oligonucleotide to a target sequence contained in the template, followed by binding of the oligonucleotide to the target, blocking access to the template and thereby regulating biosynthesis of the DNA, the RNA or the protein.
  • An additional aspect of the present invention provides pharmaceutical compositions for regulating biosynthesis of a nucleic acid or protein containing a biosynthesis regulating amount of at least one of the subject circular oligonucleotides and a pharmaceutically acceptable carrier.
  • a further aspect of the present invention provides a method of preparing a single-stranded
  • circular oligonucleotide which includes binding a linear precircle to an end-joining-oligonucleotide, joining the two ends of the precircle and recovering the circular oligonucleotide product.
  • Another aspect of the present invention provides a method of strand displacement in a double-stranded nucleic acid target by contacting the target with any one of the present circular oligonucleotides for a time and under conditions effective to denature the target and to bind the circular oligonucleotide.
  • Fig. 1A depicts the bonding patterns of
  • FIG. 1B depicts T-AT, C+GC and G-TA base triads that can form between P, target and AP nucleotides.
  • Fig. 2 schematically illustrates a circularization reaction for synthesis of single-stranded circular oligonucleotides.
  • a linear precircle oligonucleotide is bound to an oligonucleotide having the same sequence as the target, i.e. an end-joining-oligonucleotide, to form a precircle complex.
  • the circularized oligonucleotides are
  • Fig. 3 depicts the sequence of linear precursors to circular oligonucleotides, i.e. precircles (1-3 having SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7), targets (4,5 having SEQ ID NO: 8 and SEQ ID NO: 9), circular oligonucleotides (6,7,8 and 13 having SEQ ID NO: 5-7 and 14), and linear oligonucleotides (9-12 and 14 having SEQ ID NO: 10-13 and 15) described in the Examples.
  • precircles 1-3 having SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7
  • targets 4,5 having SEQ ID NO: 8 and SEQ ID NO: 9
  • circular oligonucleotides (6,7,8 and 13 having SEQ ID NO: 5-7 and 14
  • linear oligonucleotides 9-12 and 14 having SEQ ID NO: 10-13 and 15
  • Fig. 4 depicts the structure of a linear precircle complexed with an end-joining-oligonucleotide before ligation.
  • Fig. 5 depicts the effect of pH on circular oligonucleotide:target complex formation as measured by Tm. Filled circles represent the stability at different pH values for a 6:4 complex while filled squares depict the stability of a 7:5 complex.
  • the sequences of circular oligonucleotides 6 and 7 and targets 4 and 5 are presented in Fig. 3.
  • Fig. 6A depicts the effect of loop size on complex formation, with a comparison between binding to two targets: a simple (dA) 12 target (squares) and a 36 nucleotide oligonucleotide target (circles).
  • Fig. 6B depicts the effect of target and binding domain length on complex formation.
  • Fig. 7 depicts a complex formed between a circular oligonucleotide and a target where the P and AP binding domains are staggered on the target.
  • Fig. 8 depicts replacement of one strand of a fluorecently labeled double stranded target (SEQ ID NO: 11) by either a linear oligonucleotide having SEQ ID NO: 8 (dotted line) or a circular oligonucleotide having SEQ ID NO: 5 (solid line). Strand replacement was measured by an increase in fluorescein fluorescence intensity (Y-axis) as a function of time (X-axis).
  • Fig. 9 depicts a plot of observed pseudo-first order rate constant, K obs for duplex target (SEQ ID NO: 5) at several concentrations. Uncertainty in rate constants are no more than ⁇ 10%. The depicted curve is a rectangular hyperbola generated as a best fit. A double reciprocal plot of the data, i.e., [circular oligonucleotide] -1 vs (K obs ) -1 is linear with a slope of 8.95 ⁇ 10 -6 sec.M -1 and a y-intercept of 39.8 sec.
  • the present invention relates to single-stranded circular oligonucleotides, i.e. circles, which can bind to nucleic acid targets with higher affinity and selectivity than a corresponding linear
  • both single- and double-stranded nucleic acids can be targets for binding by the present circular oligonucleotides.
  • the strong, selective binding of these circles to either single- or double-stranded targets provides a variety of uses, including methods of regulating such biological processes as DNA replication, RNA transcription, RNA splicing and processing, protein translation and the like.
  • the ability of these circles to dissociate double-stranded nucleic acids and to selectively and stably bind to targeted nucleic acids makes them ideal as diagnostic probes or as markers to localize, for example, specific sites in a chromosome or other DNA or RNA molecules.
  • the present circles are useful for isolation of
  • the single-stranded circular oligonucleotides of the present invention have at least one parallel binding (P) domain and at least one anti-parallel binding (AP) domain and have a loop domain between each binding domain, so that a circular
  • each P and AP domain exhibits sufficient complementarity to bind to one strand of a defined nucleic acid target with the P domain binding to the target in a parallel manner and the AP domain binding to the target in an anti-parallel manner.
  • the schematic illustration set forth below shows the circular arrangement of one set of P and AP oligonucleotide domains relative to each other as well as when bound to a target (T, as indicated below).
  • binding of nucleic acids in a parallel manner means that the 5' to 3' orientation is the same for each strand or nucleotide in the complex. This is the type of binding present between the target and the P domain.
  • binding of nucleic acids in an anti-parallel manner means that the 5' to 3' orientations of two strands or nucleotides in a complex lie in opposite directions, i.e. the strands are aligned as found in the typical Watson-Crick base pairing arrangement of double helical DNA.
  • binding domains are separated from other P and AP domains by loop domains whose lengths are
  • a loop domain of a circular oligonucleotide bound to a given target can be an AP or P domain for binding to a second target when the circular oligonucleotide releases from the first target.
  • the nucle ⁇ tide sequences of the P and AP domains can be determined from the defined sequence of the nucleic acid target by reference to the base pairing rules provided hereinbelow.
  • a target can be either single- or double-stranded and is selected by its known functional and structural characteristics.
  • some preferred targets can be coding regions, origins of replication, reverse transcriptase binding sites, transcription regulatory elements, RNA splicing junctions, or ribosome binding sites, among others.
  • a target can also be selected by its capability for detection or isolation of a DNA or RNA template.
  • Preferred targets are rich in purines, i.e. in adenines and guanines.
  • RNA can be known in full or in part.
  • the sequences of the P and AP domains are designed with the necessary degree of complementarity to achieve binding, as detected by known procedures, for example by a change in light absorption or fluorescence.
  • the target sequence can be represented by a consensus sequence or be only partially known.
  • circular oligonucleotides (circles) which bind to an entire class of targets represented by a consensus sequence can be provided by designing the P and AP domains from the target consensus sequence. In this instance some of the targets may match the consensus sequence exactly and others may have a few mismatched bases, but not enough mismatch to prevent binding.
  • a portion of a target sequence is known, one skilled in the art can refer to the base pairing rules provided hereinbelow to design circles which bind to that target with higher affinity than a linear oligonucleotide that has a sequence corresponding to that of the circle.
  • the present invention is also directed to circles having P and AP domains which are
  • nucleic acid target wherein a sufficient number, but not necessarily all, nucleotide positions in the P and AP domains are determined from the target sequence in accordance with the base pairing rules of this invention.
  • the number of determined (i.e. known) positions is that number of positions which are necessary to provide sufficient complementarity for binding of the subject
  • oligonucleotides to their targets, as detected by standard procedures including a change in light
  • the base pairing rules of the present invention provide for the P domain to bind to the target by forming base pairs wherein the P domain and target nucleotides have the same 5' to 3' orientation. In particular, these rules are satisfied to the extent needed to achieve binding of a circular oligonucleotide to its nucleic acid target, i.e. the degree of
  • P when a base for a position in the target is thymine, or a thymine analog, then P has cytosine or guanine, or suitable analogs thereof, in a corresponding position;
  • P when a base for a position in the target is cytosine, or a cytosine analog, then P has cytosine, thymine or uracil, or suitable analogs thereof, in a corresponding position;
  • P when a base for a position in the target is uracil, or a uracil analog, then P has cytosine, guanine, thymine, or uracil, or suitable analogs
  • the base pairing rules of the present invention provide for the AP domain to bind to the target by forming base pairs wherein the AP domain and target nucleotides are oriented in opposite directions.
  • these rules are satisfied to the extent necessary to achieve detectable binding of a circular oligonucleotide to its nucleic acid target, i.e. the degree of complementarity can be less than 100%.
  • the base pairing rules can be adhered to only insofar as is necessary to achieve sufficient complementarity for binding to be detected between the circular
  • AP when a base for a position in the target is guanine, or a guanine analog, then AP has cytosine or uracil, or suitable analogs thereof, in a corresponding position; when a base for a position in the target is adenine, or an adenine analog, then AP has thymine or uracil, or suitable analogs thereof, in a corresponding position;
  • AP when a base for a position in the target is thymine, or a thymine analog, then AP has adenine, or a suitable analog thereof, in a corresponding position;
  • AP when a base for a position in the target is cytosine, or a cytosine analog, then AP has a guanine, or a suitable analog thereof, in corresponding position;
  • AP when a base for a position in the target is uracil, or a uracil analog, then AP has adenine or guanine, or suitable analogs thereof, in a corresponding position.
  • the P, AP and loop domains are not complementary to each other.
  • Table 1 summarizes which nucleotides can form anti-parallel base pairs or parallel base pairs with a clefined target nucleotide.
  • Two complementary single-stranded nucleic acids form a stable double helix (duplex) when the strands bind, or hybridize, to each other in the typical Watson-Crick fashion, i.e. via anti-parallel GC and AT base pairs.
  • stable duplex formation and stable triplex formation is achieved when the P and AP domains exhibit sufficient complementarity to the target sequence to achieve stable binding between the circular oligonucleotide and the target molecule.
  • Stable binding occurs when an oligonucleotide remains detectably bound to target under the required
  • complementarity between nucleic acids is the degree to which the bases in one nucleic acid strand can hydrogen bond, or base pair, with the bases in a second nucleic acid strand. Hence, complementarity can be
  • sufficient complementarity means that a sufficient number of base pairs exist between a target nucleic acid and the P and/or AP domains of the circular
  • oligonucleotide to achieve detectable binding.
  • the degree of complementarity between the P domain and the target and the AP domain and the target need not be the same.
  • the degree of complementarity between the P domain and the target and the AP domain and the target need not be the same.
  • complementarity can range from as little as about 30-40% complementarity to full, i.e. 100%, complementarity.
  • the overall degree of complementarity between the P or AP domain and the target is preferably at least about 50%.
  • the P domain can sometimes have less complementarity with the target than the AP domain has with the target, for example the P domain can have about 30% complementarity with the target while the AP domain can have substantially more complementarity, e.g. 50% to 100% complementarity.
  • the degree of complementarity that provides detectable binding between the subject circular oligonucleotides and their respective targets is dependent upon the conditions under which that binding occurs. It is well known that binding, i.e.
  • nucleic acid strands depends on factors besides the degree of mismatch between two sequences. Such factors include the GC content of the region, temperature, ionic strength, the presence of formamide and types of counter ions present. The effect that these conditions have upon binding is known to one skilled in the art. Furthermore, conditions are
  • binding means that a sufficient amount of the
  • oligonucleotide is bound or hybridized to its target to permit detection of that binding. Binding can be detected by either physical or functional properties of the target:circular oligonucleotide complex.
  • oligonucleotide can be detected by any procedure known to one skilled in the art, including both functional or physical binding assays. Binding may be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as DNA replication, RNA transcription, protein translation and the like.
  • oligonucleotides of the present invention provide additional hydrogen bonds and hence more stability since two binding domains are available for bonding to a single target nucleic acid, i.e. the P domain and the AP domain. Hence, the triplex formed by a circular target nucleic acid, i.e. the P domain and the AP domain. Hence, the triplex formed by a circular target nucleic acid, i.e. the P domain and the AP domain. Hence, the triplex formed by a circular
  • oligonucleotide bound to its target nucleic acid should melt at a higher T m than the duplex formed by a linear oligonucleotide and a target.
  • Circular oligonucleotides bind to a nucleic acid target through hydrogen bonds formed between the nucleotides of the binding domains and the target.
  • the AP domain can bind by forming Watson-Crick hydrogen bonds (Fig. 1).
  • the P domain can bind to the target nucleotides by forming non-Watson-Crick hydrogen bonds (e.g., Fig. 1 and Table I).
  • a base pair or duplex is formed.
  • a nucleotide from AP and a nucleotide from P both bind to the same target nucleotide, a base triad is formed.
  • two opposing domains of a circular oligomer form a complex with a central target, giving a triplex structure, or a triple helical complex, bounded by the two looped ends of the circle.
  • this arrangement can allow formation of up to four hydrogen bonds when two thymines bind to a target adenine and up to five hydrogen bonds when two cytosines bind to a target guanine.
  • the present circular oligonucleotides have a higher selectivity for a particular target than do corresponding linear oligonucleotides. At least two factors can contribute to this high selectivity.
  • oligonucleotides of this invention bind twice to the same central target strand. Hence two domains are involved in selecting a target. Second, protonation of cytosine in a C+G-C triad is favored only when this triad forms and the additional proton gives the triad a positive charge. This positive charge can lessen the negative charge repulsions arising from the
  • the present circular oligonucleotides can displace one strand of a double-stranded target under conditions where
  • Linear oligonucleotides do not have this capacity to displace a strand of a duplex.
  • the half-life of a double-stranded target in the presence of a complementary linear oligonucleotide is about 58 min i.e. so long that the linear oligonucleotide has little utility for displacing one strand of the duplex target.
  • a double- stranded target has a half-life of only 30 sec in the presence of the present circular oligonucleotides.
  • the circular oligonucleotides of the present invention have utility not only for binding single- stranded targets, but also for binding to double- stranded targets . Accordingly, since both single- and double-stranded nucleic acids are available as targets for the present circular oligonucleotides, these
  • circular oligonucleotides can have greater utility than linear oligonucleotides.
  • the present circular oligonucleotides are better regulators of biological processes in vivo and better in vitro
  • a P or an AP domain may bind as duplex on either side of the triple standard complex.
  • a target:circular oligonucleotide complex can be partially two stranded and partially three-stranded, wherein two-stranded portions can be P:target duplexes, without bound AP nucleotides, or AP:target duplexes, without bound P nucleotides.
  • This binding arrangement is a staggered binding arrangement.
  • Each P domain, AP domain and target can independently have about 2 to about 200 nucleotides with preferred lengths being about 4 to about 100
  • the most preferred lengths are 6 to 36 nucleotides.
  • the P and AP domains are separated by loop domains which can independently have from about 2 to about 2000 nucleotides.
  • a preferred loop length is from about 3 to about 8 nucleotides with an especially preferred length being about 5 nucleotides.
  • the loop domains do not have to be composed of nucleotide bases.
  • Non-nucleotide loops can make the present circular oligonucleotides cheaper to produce. More
  • circular oligonucleotides with non-nucleotide loops are more resistent to nucleases and therefore have a longer biological half-life than linear oligonucleotides.
  • loops having no charge, or a positive charge can be used to promote binding by eliminating negative charge repulsions between the loop and target.
  • circular oligonucleotides having uncharged or hydrophobic non-nucleotide loops can penetrate cellular membranes better than circular oligonucleotides with nucleotide loops.
  • non-nucleotide loop domains can be composed of alkyl chains, polyethylene glycol or oligoethylene glycol chains or other chains providing the necessary steric or flexibility properties which are compatible with oligonucleotide synthesis.
  • the length of these chains is equivalent to about 2 to about 2000 nucleotides, with preferred lengths
  • Preferred chains for non-nucleotide loop domains are polyethylene glycol or oligoethylene glycol chains.
  • oligoethylene glycol chains having a length similar to a 5 nucleotide chain e.g. a pentaethylene glycol, a hexaethylene glycol or a heptaethylene glycol chain, are preferred.
  • the circular oligonucleotides are single- stranded DNA or RNA, with the bases guanine (G), adenine (A), thymine (T), cytosine (C) or uracil (U) in the nucleotides, or with any nucleotide analog that is capable of hydrogen bonding in a parallel or anti-parallel manner.
  • Nucleotide analogs include
  • pseudocytidine isopseudocytidine, 3-aminophenylimidazole, 2'-O-methyl-adenosine, 7-deazadenosine, 7-deazaguanosine, 4-acetylcytidine, 5-(carboxyhydroxylmethyl)-uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thioridine, 5- carboxymethylamino-methyluridine, dihydrouridine, 2'-O-methyluridine, 2'-O-methyl-pseudouridine, beta,D-galactosylqueosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2- dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methyl
  • oligonucleotides of the present invention are unmodified G,
  • A, T, C and U nucleotides pyrimidine analogs with lower alkyl, lower alkoxy, lower alkylamine, phenyl or lower alkyl substituted phenyl groups in the 5 position of the base and purine analogs with similar groups in the 7 or 8 position of the base.
  • pyrimidine analogs with lower alkyl, lower alkoxy, lower alkylamine, phenyl or lower alkyl substituted phenyl groups in the 5 position of the base and purine analogs with similar groups in the 7 or 8 position of the base.
  • nucleotide analogs are 5-methylcytosine, 5-methyluracil,
  • lower alkyl, lower alkoxy and lower alkylamine contain from 1 to 6 carbon atoms and can be straight chain or branched. These groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, amyl, hexyl and the like.
  • a preferred alkyl group is methyl.
  • Circular oligonucleotides can be made first as linear oligonucleotides and then circularized.
  • Linear oligonucleotides can be made by any of a myriad of procedures known for making DNA or RNA oligonucleotides. For example, such procedures include enzymatic synthesis and chemical synthesis.
  • Enzymatic methods of DNA oligonucleotide synthesis frequently employ Klenow, T7, T4, Taq or E. coli DNA polymerases as described in Sambrook et al.
  • RNA oligonucleotide synthesis frequently employ SP6, T3 or T7 RNA polymerase as described in Sambrook et al. Reverse transcriptase can also be used to synthesize DNA from RNA (Sambrook et al.).
  • a template nucleic acid which can either be synthesized chemically, or be obtained as mRNA, genomic DNA, cloned genomic DNA, cloned cDNA or other recombinant DNA.
  • oligonucleotide synthesis can require an additional primer oligonucleotide which can be synthesized
  • linear oligonucleotides can be prepared by PCR techniques as described, for example, by Saiki et al., 1988, Science 239:487.
  • oligonucleotides of defined sequence can be purchased commercially or can be made by any of several different synthetic procedures including the phosphoramidite, phosphite triester, H-phosphonate and phosphotriester methods, typically by automated synthesis methods.
  • the synthesis method selected can depend on the length of the desired oligonucleotide and such choice is within the skill of the ordinary artisan.
  • the phosphoramidite and phosphite triester method produce oligonucleotides having 175 or more nucleotides while the H-phosphonate method works well for oligonucleotides of less than 100 nucleotides.
  • Synthetic, linear oligonucleotides may be purified by polyacrylamide gel electrophoresis, or by any of a number of chromatographic methods, including gel chromatography and high pressure liquid chromatography.
  • oligonucleotides may be subjected to DNA sequencing by any of the known procedures, including Maxam and Gilbert sequencing, Sanger sequencing, capillary electrophoresis sequencing the wandering spot sequencing procedure or by using selective chemical degradation of oligonucleotides bound to Hybond paper. Sequences of short
  • oligonucleotides can also be analyzed by plasma
  • the present invention provides several methods of preparing circular oligonucleotides from linear precursors (i.e. precircles), including a method wherein a precircle is synthesized and bound to an end-joining-oligonucleotide and the two ends of the precircle are joined. Any method of joining two ends of an
  • oligonucleotide is contemplated by the present
  • a simple one-step chemical method is provided to construct the subject circular oligonucleotides, or circles, from precircles.
  • An oligonucleotide is constructed which has the same sequence as the target nucleic acid; this is the end-joining oligonucleotide.
  • a DNA or RNA linear precircle is chemically or enzymatically synthesized and phosphorylated on its 5' or 3' end, again by either chemical or enzymatic means.
  • the precircle and the end- joining oligonucleotide are mixed and annealed, thereby forming a complex in which the 5' and 3' ends of the precircle are adjacent, as depicted in Fig. 2. It is preferred that the ends of the precircle fall within a binding domain, not within a loop, and preferably within the anti-parallel binding domain rather than the
  • a precircle have a 3'-phosphate rather than a 5'-phosphate.
  • the ends undergo a condensation reaction in a buffered aqueous solution containing divalent metal ions and BrCN at about pH 7.0.
  • the buffer is imidazole-Cl at pH 7.0 with a divalent metal such as Ni, Zn, Mn, or Co. Ni is the most preferred divalent metal. Condensation occurs after about 6-48 hr. of incubation at 4-37°C.
  • divalent metals such as Cu, Pb, Ca and Mg, can also be used.
  • RNA oligonucleotide incorporates the appropriate nucleotide sequences, preferably in a loop domain, into an RNA oligonucleotide to promote self splicing, since a circular product is formed under the appropriate conditions (Sugimoto et al., 1988, Biochemistry: 27: 6384-6392).
  • Enzymatic circle closure is also possible using DNA ligase or RNA ligase under conditions
  • Circular oligonucleotides can be separated from the template by denaturing gel electrophoresis or melting followed by gel electrophoresis, size selective chromatography, or other appropriate chromatographic or electrophoretic methods. The recovered circular oligonucleotides
  • oligonucleotide can be further purified by standard techniques as needed for its use in the methods of the present invention.
  • the present invention also contemplates derivatization or chemical modification of the subject oligonucleotides with chemical groups to facilitate cellular uptake.
  • covalent linkage of a cholesterol moiety to an oligonucleotide can improve cellular uptake by 5- to 10- fold which in turn improves DNA binding by about 10- fold (Boutorin et al., 1989, FEBS Letters 254: 129-132).
  • Other ligands for cellular receptors may also have utility for improving cellular uptake, including, e.g. insulin, transferrin and others.
  • derivatization of oligonucleotides with poly-L-lysine can aid oligonucleotide uptake by cells
  • Certain protein carriers can also facilitate cellular uptake of oligonucleotides, including, for example, serum albumin, nuclear proteins possessing signals for transport to the nucleus, and viral or bacterial proteins capable of cell membrane penetration. Therefore, protein carriers are useful when associated with or linked to the circular oligonucleotides of this invention. Accordingly, the present invention
  • oligonucleotides with groups capable of facilitating cellular uptake, including hydrocarbons and non-polar groups, cholesterol, poly-L-lysine and proteins, as well as other aryl or steroid groups and polycations having analogous beneficial effects, such as phenyl or naphthyl groups, quinoline, anthracene or phenanthracene groups, fatty acids, fatty alcohols and sesquiterpenes,
  • the present invention further contemplates derivatization of the subject oligonucleotides with agents that can cleave or modify the target nucleic acid or other nucleic acid strands associated with or in the vicinity of the target.
  • agents that can cleave or modify the target nucleic acid or other nucleic acid strands associated with or in the vicinity of the target.
  • viral DNA or RNA can be targeted for destruction without harming cellular nucleic acids by administering a circular
  • Nucleic acid destroying agents that are contemplated by the present invention as having cleavage or modifying activities include, for example, RNA and DNA nucleases, ribozymes that can cleave RNA, azidoproflavine,
  • oligonucleotides that can be adapted for use with the subject circular oligonucleotides.
  • Derivatization of the subject circular oligonucleotides with groups that facilitate cellular uptake or target binding, as well as derivatization with nucleic acid destroying agents or drugs can be done by any of the procedures known to one skilled in the art.
  • the desired groups can be added to nucleotides before synthesis of the oligonucleotide.
  • these groups can be linked to the 5-position of T or C and these modified T and C nucleotides can be used for synthesis of the present circular oligonucleotides.
  • derivatization of selected nucleotides permits incorporation of the group into selected domains of the circular oligonucleotide. For example, in some
  • modification in the phosphodiester backbone of circular oligonucleotides is also contemplated.
  • modifications can aid uptake of the oligonucleotide by cells or can extend the biological half-life of such nucleotides.
  • circular oligonucleotides may penetrate the cell membrane more readily if the negative charge on the internucleotide phosphate is eliminated. This can be done by replacing the negatively charged phosphate oxygen with a methyl group, an amine or by changing the phosphodiester linkage into a
  • phosphotriester linkage by addition of an alkyl group to the negatively charged phosphate oxygen.
  • one or more of the phosphate atoms which is part of the normal phosphodiester linkage can be replaced.
  • NH-P, CH 2 -P or S-P linkages can be formed.
  • the present invention contemplates using methylphosphonates, phosphorothioates,
  • phosphorodithioates phosphotriesters and phosphorusboron (Sood et al., 1990, J. Am. Chem. Soc. 112: 9000) linkages.
  • the phosphodiester group can be replaced with siloxane, carbonate, acetamidate or thioether groups. These modifications can also increase the resistance of the subject oligonucleotides to nucleases. Methods for synthesis of oligonucleotides with modified
  • Circular oligonucleotides with non-nucleotide loops can be prepared by any known procedure.
  • Durand et al. (1990, Nucleic Acids Res. 18: 6353-6359) provides synthetic procedures for linking non-nucleotide chains to DNA. Such procedures can generally be adapted to permit an automated synthesis of a linear oligonucleotide precursor which is then used to make a circular oligonucleotide of the present
  • groups reactive with nucleotides in standard DNA synthesis e.g. phosphoramidite, H-phosphonate, dimethoxytrityl, monomethoxytrityl and the like, can be placed at the ends of non-nucleotide chains and nucleotides corresponding to the ends of P and AP domains can be linked thereto.
  • RNA oligonucleotides can be used since RNA:DNA hybrids are more stable than DNA:DNA hybrids. Additional binding stability can also be provided by using 2'-O-methyl ribose in the present circular oligonucleotides. Phosphoramidite chemistry can be used to synthesize RNA oligonucleotides as described (Reese, C. B. In Nucleic Acids & Molecular Biology; Springer-Verlag: Berlin, 1989; Vol. 3, p. 164; and Rao, et al., 1987, Tetrahedron Lett. 28: 4897).
  • RNA 2'-O-methyloligoribonucleo-tides differ only slightly.
  • RNA 2'-O-methyloligonucleotides can be prepared with minor modifications of the amidite, H- phosphonate or phosphotriester methods (Shibahara et al, 1987, Nucleic Acids Res. 15: 4403; Shibahara et al., 1989, Nucleic Acids Res. 17: 239; Anoue et al., 1987, Nucleic Acids Res. 15: 6131).
  • circular oligonucleotides can accelerate the reaction
  • the double-stranded nucleic acid target does not have to be subjected to denaturing conditions before binding of the present circular oligonucleotides.
  • the circular oligonucleotides can bind to both single- and double-stranded nucleic acid targets under a wider variety of conditions, and particularly under
  • oligonucleotides are several orders of magnitude faster at accelerating duplex nucleic acid strand displacement than are the corresponding linear oligonucleotides.
  • the present invention therefore provides a means to displace one strand of a double-stranded nucleic acid target with one of the subject circular oligonucleotides without the necessity of prior
  • the present invention provides a method of strand displacement in a double-stranded nucleic acid target by contacting the target with one of the subject circular oligonucleotides for a time and under conditions
  • the target for the present circular oligonucleotides can be a double-stranded nucleic acid, either RNA or DNA, which has not undergone denaturation by, for example, heating or exposure to alkaline pH.
  • the nucleic acids for strand displacement can be present in an organism or present in a sample which includes an impure or pure nucleic acid preparation, a tissue section, a prokaryoti ⁇ or
  • nucleic acid targets for strand displacement by the present circular oligonucleotides include viral, bacterial, fungal or mammalian nucleic acids.
  • conditions effective to denature the target by strand displacement and thereby permit binding include having a suitable circular oligonucleotide to target nucleic acid ratio.
  • a suitable ratio of circular oligonucleotide to target is about 1 to about 100, and is preferably about 1 to about 50.
  • a time effective to denature a double-stranded nucleic acid by strand-displacement with an oligonucleotide of the present invention is about 1 minute to about 16 hours.
  • a circular oligonucleotide can associate with a duplex target by first binding in the P domain. Such P domain binding juxtaposes the AP domain nucleotides to compete for Watson-Crick binding to target nucleotide. This P domain pre-association followed by AP domain nucleotide competition for Watson-Crick binding may form the basis for the observed acceleration in strand displacement by circular oligonucleotides.
  • the subject circular oligonucleotides have three important features which enable duplex strand displacement. First, the circular oligonucleotide has the ability to preassociate, which results in a high local concentration. Second, the circular oligonucleotide contains a second (AP) binding domain, which competes for binding to a complementary strand of the duplex. Finally, the circular oligonucleotide has the ability to preassociate, which results in a high local concentration. Second, the circular oligonucleotide contains a second (AP) binding domain, which competes for binding
  • oligonucleotide binds with higher affinity than the displaced strand of the duplex, thereby driving the reaction to completion.
  • the present invention contemplates a variety of utilities for the subject circular oligonucleotides which are made possible by their selective and stable binding properties with both single- and double-stranded targets. Some utilities include, but are not limited to: use of circular oligonucleotides of defined
  • oligonucleotides to provide sequence specific stop signals during polymerase chain reaction (PCR); covalent attachment of a drug, drug analog or other therapeutic agent to circular oligonucleotides to allow cell type specific drug delivery; labeling circular oligonucleotides with a detectable reporter molecule for localizing, quantitating or identifying complementary target nucleic acids; and binding circular oligonucleotides to provide sequence specific stop signals during polymerase chain reaction (PCR); covalent attachment of a drug, drug analog or other therapeutic agent to circular oligonucleotides to allow cell type specific drug delivery; labeling circular oligonucleotides with a detectable reporter molecule for localizing, quantitating or identifying complementary target nucleic acids; and binding circular oligonucleotides to provide sequence specific stop signals during polymerase chain reaction (PCR); covalent attachment of a drug, drug analog or other therapeutic agent to circular oligonucleotides to allow cell type specific drug delivery; labeling circular oligonucleotides
  • oligonucleotides to a cellular or viral nucleic acid template and regulating biosynthesis directed by that template.
  • the subject circular oligonucleotides can be attached to a solid support such as silica, cellulose, nylon, and other natural or synthetic materials that are used to make beads, filters, and column chromatography resins. Attachment procedures for nucleic acids to solid supports of these types are well known; any known attachment procedure is contemplated by the present invention. A circular oligonucleotride attached to a solid support can then be used to isolate a
  • complementary nucleic acid can be done by incorporating the oligonucleotide:solid support into a column for chromatographic procedures. Other isolation methods can be done without incorporation of the
  • oligonucleotide solid support into a column, e.g. by utilization of filtration procedures.
  • oligonucleotide:solid supports can be used, for example, to isolate poly(A) mRNA from total cellular or viral RNA by making a circular oligonucleotide with P and AP domain poly(dT) or poly(U) sequences. Circular
  • oligonucleotides are ideally suited to applications of this type because they are nuclease resistant and bind target nucleic acids so strongly.
  • PCR polymerase chain reaction
  • the present invention also contemplates using the subject circular oligonucleotides for targeting drugs to specific cell types.
  • Such targeting can allow selective destruction or enhancement of particular cell types, e.g. inhibition of tumor cell growth can be attained.
  • Different cell types express different genes, so that the concentration of a particular mRNA can be greater in one cell type relative to another cell type, such an mRNA is a target mRNA for cell type specific drug delivery by circular oligonucleotides linked to drugs or drug analogs. Cells with high concentrations of target mRNA are targeted for drug delivery by
  • the present invention also contemplates labeling the subject circular oligonucleotides for use as probes to detect a target nucleic acid.
  • Labelled circular oligonucleotide probes have utility in
  • Circular oligonucleotide probes of this invention represent a substantial improvement over linear nucleic acid probes because the circular oligonucleotides can replace one strand of a double-stranded nucleic acid, and because the present oligonucleotides have two binding domains which not only provide increased binding stability but also impart a greater sequence selectivity (or specificity) for the target: oligonucleotide
  • Labeling of a circular oligonucleotide can be done by incorporating nucleotides linked to a "reporter molecule" into the subject circular oligonucleotides.
  • a “reporter molecule”, as defined herein, is a molecule or atom which,, by its chemical nature, provides an
  • Detection can be either qualitative or quantitative.
  • the present invention contemplates using any commonly used reporter molecule including
  • radionuclides enzymes, biotins, ps ⁇ ralens,
  • reporter molecules fluorophores, chelated heavy metals, and luciferin.
  • the most commonly used reporter molecules are either
  • enzymes fluorophores or radionuclides linked to the nucleotides which are used in circular oligonucleotide synthesis.
  • Commonly used enzymes include horseradish peroxidase, alkaline phosphatase, glucose oxidase and ß-galactosidase, among others.
  • the substrates to be used with the specific enzymes are generally chosen because a detectably colored product is formed by the enzyme acting upon the substrate.
  • p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for horseradish peroxidase, 1,2-phenylenediamine, 5-aminosalicyclic acid or toluidine are commonly used.
  • the probes so generated have utility in the detection of a specific DNA or RNA target in, for example, Southern analysis, Northern analysis, in situ hybridization to tissue sections or chromosomal squashes and other analytical and diagnostic procedures.
  • the methods of using such hybridization probes are well known and some examples of such methodology are provided by Sambrook et al.
  • the present circular oligonucleotides can be used in conjunction with any known detection or
  • the present circular oligonucleotides can be used in any hybridization procedure which quantitates a target nucleic acid, e.g., by competitive hybridization between a target nucleic acid present in a sample and a labeled tracer target for one of the present oligonucleotides.
  • the reagents needed for making a circular oligonucleotide probe and for utilizing such a probe in a hybridization procedure can be marketed in a kit.
  • the kit can be compartmentalized for ease of utility and can contain at least one first container providing reagents for making a precircle precursor for a circular oligonucleotide, at least one second
  • container providing reagents for labeling the precircle with a reporter molecule, at least one third container providing regents for circularizing the precircle, and at least one fourth container providing reagents for isolating the labeled circular oligonucleotide.
  • kits for isolation of a template nucleic acid has at least one first container providing a circular oligonucleotide which is complementary to a target contained within the template.
  • a kit for isolation of a template nucleic acid has at least one first container providing a circular oligonucleotide which is complementary to a target contained within the template.
  • template nucleic acid can be cellular and/or viralpoly(A) mRNA and the target can be the poly(A) tail.
  • circular oligonucleotides of the present invention which have utility for isolation of poly(A)+ mRNA have p and AP domain sequences of poly(dT) or poly(U).
  • kits useful when diagnosis of a disease depends upon detection of a specific, known target nucleic acid.
  • nucleic acid targets can be, for example, a viral nucleic acid, an extra or missing chromosome or gene, a mutant cellular gene or chromosome, an aberrantly expressed RNA and others.
  • the kits can be
  • one aspect of the present invention provides a method of regulating biosynthesis of a DNA, an RNA or a protein by contacting at least one of the subject circular oligonucleotides with a nucleic acid template for that DNA, that RNA or that protein in an amount and under conditions sufficient to permit the binding of the oligonucleotide(s) to a target sequence contained in the template.
  • the binding between the oligonucleotide(s) and the target blocks access to the template, and thereby regulates biosynthesis of the nucleic acid or the protein. Blocking access to the template prevents proteins and nucleic acids involved in the biosynthetic process from binding to the template, from moving along the template, or from recognizing signals encoded within the template.
  • RNA templates bound by the subject circular oligonucleotides are susceptible to degradation by RNase H and RNase H degradation of a selected RNA template can thereby regulate use of the template in biosynthetic processes.
  • biosynthesis of a nucleic acid or a protein includes cellular and viral processes such as DNA replication, DNA reverse transcription, RNA transcrip-tion, RNA splicing, RNA polyadenylation, RNA transloca-tion and protein translation, and of which can lead to production of DNA, RNA or protein, and involve a nucleic acid template at some stage of the biosynthetic process.
  • regulating biosynthesis includes inhibiting, stopping, increasing, accelerating or delaying biosynthesis. Regulation may be direct or indirect, i.e. biosynthesis of a DNA, RNA or protein may be regulated directly by binding a circular
  • oligonucleotide to the template for that DNA, RNA or protein; alternatively, biosynthesis may be regulated indirectly by oligonucleotide binding to a second template encoding a protein that plays a role in
  • the nucleic acid templates can be RNA or DNA and can be single-stranded or double-stranded. While the present circular oligonucleotides bind to only one strand of a target present in the template, double-stranded templates are opened during biosynthetic processes and thereby become available for binding.
  • the P domain of the present circular oligonucleotides can bind to a double-stranded target and place AP domain nucleotides in a position to compete for Watson-Crick binding to target nucleotides.
  • DNA replication from a DNA template is mediated by proteins which bind to an origin of
  • circular oligonucleotides are selected which bind to one or more targets in an origin of replication. Such binding blocks template access to proteins involved in DNA replication. Therefore initiation and procession of DNA replication is inhibited.
  • expression of the proteins which mediate DNA replication can be inhibited at, for example, the transcriptional or translational level.
  • DNA replication from an RNA template is mediated by reverse transcriptase binding to a region of RNA also bound by a nucleic acid primer.
  • reverse transcriptase or primer binding can be blocked by binding a circular oligonucleotide to the primer binding site, and thereby blocking access to that site.
  • inhibition of DNA replication can occur by binding a circular oligonucleotide to the primer binding site, and thereby blocking access to that site.
  • inhibition of DNA replication can occur by binding a circular
  • oligonucleotide to a site residing in the RNA template since such binding can block access to that site and to downstream sites, i.e. sites on the 3' side of the target or binding site.
  • RNA polymerase recognizes and binds to specific start sequences, or promoters, on a DNA template. Binding of RNA polymerase opens the DNA template.
  • transcriptional regulatory elements include enhancer sequences, upstream activating sequences, repressor binding sites and others. All such promoter and transcriptional regulatory elements, singly or in combination, are targets for the subject circular oligonucleotides. Oligonucleotide binding to these sites can block RNA polymerase and transcription factors from gaining access to the template and thereby
  • RNA especially mRNA and tRNA.
  • oligonucleotides can be targeted to the coding region or 3'-untranslated region of the DNA template to cause premature termination of transcription.
  • One skilled in the art can readily design oligonucleotides for the above target sequences from the known sequence of these regulatory elements, from coding region sequences, and from consensus sequences.
  • RNA transcription can be increased by, for example, binding a circular oligonucleotide to a
  • Negative transcriptional regulatory elements include repressor sites or operator sites, wherein a repressor protein binds and blocks
  • Oligonucleotide binding to repressor or operator sites can block access of repressor proteins to their binding sites and thereby increase transcription.
  • the primary RNA transcript made in eukaryotic cells, or pre-mRNA, is subject to a number of
  • introns are removed from the pre-mRNA in splicing reactions.
  • the 5' end of the mRNA is modified to form the 5' cap structure, thereby stabilizing the mRNA.
  • oligonucleotides can be used to block any of these processes.
  • a pre-mRNA template is spliced in the nucleus by ribonucleoproteins which bind to splice junctions and intron branch point sequences in the pre-mRNA.
  • Consensus sequences for 5' and 3' splice junctions and for the intron branch point are known.
  • inhibition of ribonucleoprotein binding to the splice junctions or inhibition of covalent linkage of the 5' end of the intron to the intron branch point can block splicing.
  • Maturation of a pre-mRNA template can, therefore, be blocked by preventing access to these sites, i.e. by binding circular oligonucleotides of this invention to a 5' splice junction, an intron branch point or a 3' splice junction.
  • Splicing of a specific pre-mRNA template can be inhibited by using circular oligonucleotides with sequences that are complementary to the specific pre-mRNA splice junction(s) or intron branch point.
  • a collection of related splicing of pre-mRNA templates can be inhibited by using a mixture of circular oligonucleotides having a variety of sequences that, taken together, are
  • Polyadenylation involves recognition and cleavage of a pre-mRNA by a specific RNA endonuclease at specific polyadenylation sites, followed by addition of a poly(A) tail onto the 3' end of the pre-mRNA. Hence, any of these steps can be inhibited by binding the subject oligonucleotides to the appropriate site.
  • RNA translocation from the nucleus to the cytoplasm of eukaryotic cells appears to require a poly(A) tail.
  • a circular oligonucleotide is designed in accordance with this invention to bind to the poly(A) tail and thereby block access to the poly (A) tail and inhibit RNA translocation.
  • both the P and AP domains can consist of about 10 to about 50 thymine residues, and preferably about 20 residues.
  • Especially preferred P and AP domain lengths for such an oligonucleotide are about 6 to about 12 thymine residues.
  • Protein biosynthesis begins with the binding of ribosomes to an mRNA template, followed by initiation and elongation of the amino acid chain via translational "reading" of the mRNA. Protein biosynthesis, or
  • targets in the template mRNA include the ribosome binding site (Shine-Delgarno sequence), the 5' mRNA cap site, the initiation codon, and sites in the protein coding sequence.
  • targets contemplated by this invention include the ribosome binding site (Shine-Delgarno sequence), the 5' mRNA cap site, the initiation codon, and sites in the protein coding sequence.
  • proteins which share domains of nucleotide sequence homology. Thus, inhibition of protein biosynthesis for such a class can be
  • genetic disorders can be corrected by inhibiting the production of mutant or over-produced proteins, or by increasing production of an under-expressed proteins; the expression of genes encoding factors that regulate cell proliferation can be inhibited to control the spread of cancer; and virally encoded functions can be inhibited to combat viral infection.
  • Some types of genetic disorders that can be treated by the circular oligonucleotides of the present invention include Alzheimer's disease, some types of arthritis, sickle cell anemia and others. Many types of viral infections can be treated by utilizing the
  • circular oligonucleotides of the present invention including infections caused by influenza, rhinovirus, HIV, herpes simplex, papilloma virus, cytomegalovirus, Epstein-Barr virus, adenovirus, vesticular stomatitus virus, rotavirus and respitory synctitial virus among others.
  • animal and plant viral infections may also be treated by
  • the c-myc gene is one example of a gene which can have a role in cell proliferation. Inhibition of c-myc expression has been demonstrated in vitro using a linear oligonucleotide complementary to a target 115 bp upstream of the c-myc transcription start site (Cooney et al., 1988, Science 241: 456-459). Circular
  • oligonucleotides of SEQ ID NO:1, and SEQ ID NO: 2, as depicted below, are complementary to the c-myc promoter at nucleotides -131 to -120 and -75 to -62, respectively, and are provided to inhibit c-myc
  • N can be any nucleotide or nucleotide analog.
  • HIV Human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • the retroviral genome is transcribed as a single, long transcript, part of which is spliced to yield RNA encoding viral envelope proteins. Inhibition of HIV infection can be
  • oligonucleotides to bind to a number of regions within the HIV genome, including coding regions for functions that replicate the genome (i.e., the pol or reverse transcriptase function) or functions that control gene expression (e.g. the tat, rev or other functions).
  • functions that replicate the genome i.e., the pol or reverse transcriptase function
  • functions that control gene expression e.g. the tat, rev or other functions.
  • splice sites, poly(A) addition signals, cap or initiator codon sites, and sites implicated in ribosome assembly can be
  • the terminal structures of the retroviral genome are also excellent targets for
  • the present invention provides two circular oligonucleotides, set forth in SEQ ID NO: 3 and SEQ ID NO: 4 wherein N is any nucleotide or
  • SEQ ID NO: 3 is complementary to an HIV-1 splice junction (nucleotides 6039-52), while SEQ ID NO:4 is complementary to part of the tat gene (nucleotides 5974-88).
  • the circular form of SEQ ID NO: 3 is depicted below, wherein nucleotide number 1 is the first
  • nucleotide in the P domain i.e., the first T on the top line corresponds to base 1.
  • SEQ ID NO: 4 The circular form of SEQ ID NO: 4 is depicted below wherein nucleotide number 1 is the first nucleotide of the P domain.
  • SEQ ID NO: 4 can inhibit HIV infection both in vitro and in vivo. In vitro screening for circular
  • oligonucleotide effectiveness against HIV infection permits one skilled in the art to judge the stability of oligonucleotide: target binding and to assess in vivo efficacy and binding stability.
  • circular oligonucleotides can be added to the growth medium of an appropriate cell line infected with
  • oligonucleotides or circular oligonucleotides can be added at the time of infection or after HIV infection.
  • Addition before or after infection allows assessment of whether the subject oligonucleotide can prevent or simply inhibit HIV infection respectively.
  • the extent of inhibition of HIV infection or replication can be judged by any of several assay systems, including assessment of the proportion of oligonucleotide-treated cells surviving after infection relative to survival of untreated cells, assessment of the number of syn ⁇ ytia formed in treated and untreated
  • HIV infected cells determination of the amount of viral antigen produced in treated and untreated cells.
  • human volunteers with AIDS or ARC can be administered with the subject circular oligonucleotides since the oligonucleotides do not appear to be
  • cytotoxic The disease status of these volunteers can then be assessed to determine the efficacy of the subject oligonucleotides in treating and preventing AIDS infection.
  • a further aspect of this invention provides pharmaceutical compositions containing the subject circular oligonucleotides with a pharmaceutically acceptable carrier.
  • the subject circular oligonucleotides with a pharmaceutically acceptable carrier.
  • oligonucleotides are provided in a therapeutically effective amount of about 0.1 ⁇ g to about 100 mg per kg of body weight per day, and preferably of about 0.1 ⁇ g to about 10 mg per kg of body weight per day, to bind to a nucleic acid in accordance with the methods of this invention. Dosages can be readily determined by one of ordinary skill in the art and formulated into the subject pharmaceutical compositions.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • solvents dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is
  • compositions incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • the subject oligonucleotides may be administered topically or parenterally by, for example, intraveneous, intramuscular, intraperitoneal
  • the subject oligonucleotides may be orally administered.
  • the subject oligonucleotides may be incorporated into a cream, solution or suspension for topical administration.
  • oligonucleotides may be protected by enclosure in a gelatin capsule.
  • Oligonucleotides may be incorporated into liposomes or lipsomes modified with polyethylene glycol for parenteral administration. Incorporation of additional substances into the liposome, for example, antibodies reactive against membrane proteins found on specific target cells, can help target the
  • End Joining Oligonucleotide According to the present invention, a simple one-step chemical method has been developed to construct circles from linear precursors (precircles). A DNA oligonucleotide was constructed which had the same sequence as the eventual target, this is the end-joining-oligonucleotide. A precircle oligonucleotide was then constructed and chemically phosphorylated on the 5'-end or 3'-end. As depicted in Fig. 2 , the precircle and end-joining-oligonucleotide were mixed and allowed to form a complex in which the ends were
  • Closure of a circle in the AP domain was superior to closure in the P domain.
  • BrCN/imidazole/NiCl 2 was used under the established optimal conditions except that ligation efficiency was observed at both 4°C and 25°C.
  • EDC wasused at 200 mM with 20 mM MgCl 2 , 50 mM MES (pH 6.0) at 4°C or 25°C with incubation for 4 days.
  • Circularization reactions were performed using a dA 12 end-joining-oligonucleotide and the established optimal conditions, except that 5 nmoles of precircle and end-joining-oligonucleotide were used. Products were visualized under UV light after separation by denaturing gel electrophoresis.
  • circle 6 having the same sequence as precircle 1, bound to target 4 with a T of 57.5°C and a free energy of binding that was 8.6 kcal/mol more favorable than the corresponding Watson-Crick duplex.
  • the binding affinity of circle 6 for an RNA target was tested by synthesizing oligoribonucleotide rA 12 and determining the T m of circle 6 with rA 12 .
  • the T m of circle 6 with rA 12 was 58.3°C compared with 57.8°C with dA 12 .
  • the data indicate that circles bind to RNA targets as strongly or more strongly than as to DNA targets.
  • oligonucleotides with one variable base was constructed. Binding energies for a circle complexed with these targets were measured; the selectivity was defined by the free energy difference between the correct sequence and mismatched sequences. The selectivity obtained with the circular structure was then directly compared to the selectivity of an analogous linear oligonucleotide.
  • Circular oligonucleotide 8 was prepared from a linear precircle having SEQ ID NO: 7:
  • 5' -pTCTTTCCACACCTTTCTTTTCTTCACACTTCTTT was cyclized by assembly around an end-joining oligonucleotide having the sequence 5' -AAGAAAAGAAAG (SEQ ID NO: 9) using BrCN/imidazole to close the final bond, as described in Example 1.
  • the circular structure was confirmed by its resistance to a 3'-exonuclease and 5'-phosphatase.
  • Table III displays the results of the mismatch experiments.
  • Experiments 1-4 show the effects of a T-X target mismatch on a DNA duplex.
  • target stranded complexes
  • experiments 9-12 give the effects of a C-Y mismatch on the two stranded duplex.
  • the circular ligand shows greater selectivity for its correctly matched sequence than does the standard linear oligomer.
  • the selectivity advantage ranges from 1.3 to 2.2 kcal/mol for the C-Y-C series to 3.0 to 3.4 kcal/mol for the T-X-T series.
  • deoxycytosine The addition of this positive charge may lessen the negative charge repulsions arising from the high density of phosphates in the complex and thereby increase binding stability.
  • circular oligonucleotides as described herein, to have both higher binding affinity and higher selectivity than can be achieved with Watson-Crick duplexes alone.
  • Precircle linear oligonucleotides similar to precircle 1 were synthesized with 2, 3, 4, 5, 6 and 10 base loops using an arbitrary sequence of alternating C and A residues. Each of these precircles was designed to bind to the A 12 template (i.e. target 4 (SEQ ID NO: 8)).
  • target 4 SEQ ID NO: 8
  • circles with 4, 5, 6 and 10 base loops showed that a five-nucleotide loop size was optimum for the circle binding either to template A 12 or to a longer 36mer sequence containing the A 12 binding site (see Fig. 6A).
  • Fig. 6B illustrates that considerably higher T m 's were observed for circle: target complexes relative to Watson-Crick duplexes having the same length as the binding domains (determined in 0.1 M NaCl, pH 7).
  • T m the binding domains
  • a 12-base circular complex melted at about the same temperature as a 24-base duplex.
  • the 4-base circular complex melted at 34°C, whereas the corresponding Watson-Crick duplex T m was less than 0°C.
  • target complexes two analogs of circle 7 (having SEQ ID NO: 6) were synthesized.
  • the six C's in the binding domains were methylated leaving the loop unmethylated (Me 6 ).
  • all twelve C's were methylated (Me 12 ). Melting temperatures for the complexes of these methylated circle with target
  • the Me 6 complex had a T m of 71.1°C (compared to 61.8°C for the unmethylated circle), and the
  • Me 12 circle had a T m of 72.4°C.
  • use of the natural base m 5 C in place of C increased stability substantially, and in one case resulted in a 12-base complex which melted 10.6°C higher than an unmethylated circle and 28.6°C higher than the corresponding unmethylated Watson-Crick duplex.
  • loop domains of circular oligonucleotides were replaced with polyethylene or oligoethylene glycol chains of different lengths and the effect of such synthetic loops upon circular oligonucleotide binding and nuclease resistance was assessed.
  • Circular oligonucleotides were synthesized having tetra-, penta-, or hexa-ethylene glycol chain loop domains.
  • the ethylene glycol chain was synthetically prepared for automated DNA synthetic procedures using the method of Durand et al. (1990, Nucleic Acids Res. 18: 6353-6359). Briefly, a
  • phosphoramidite was placed on a hydroxy group at one end of the ethylene glycol chain and a dimethoxytrityl (DMT) moiety was placed on the other terminal ethylene glycol hydroxy group.
  • DMT dimethoxytrityl
  • Circularization steps were performed by procedures described in Example 1.
  • a linear oligonucleotide precircle having a tetraethylene loop domain was not efficiently circularized. This result indicates that a tetraethylene loop domain may be too short for optimal binding to a target.
  • Target I was a 12-base oligonucleotide having no non-target nucleotides and Target II was a 36-base oligonucleotide having a 12-base target within it.
  • the target sequences utilized were 5'-AAGAAAAGAAAG-3' (SEQ ID NO: 9) and 5'-AAAAAAAAAAAA-3' (SEQ ID NO: 8), the latter is termed a poly(dA) 12 target sequence.
  • Tm melting temperatures
  • CACAC nucleotide loop sequence and a poly(dT) 12 sequence for both P and AP domains was 57.8°C when bound to a poly
  • the Tm value observed for a circular oligonucleotide having a HEG loop is about 4.5°C higher than that of a circular oligonucleotide with a PEG loop. Therefore, circular oligonucleotides with hexaethylene glycol loop domains bind with greater stability than do circular oligonucleotides with tetra- or penta-ethylene glycol loops.
  • Circular oligonucleotides were tested for nuclease resistance when unbound and when bound to a target oligonucleotide. All circular oligonucleotides, whether bound or unbound, were completely resistant to exonucleases. Endonuclease sensitivity was assessed using S1 nuclease according to the manufacturer's suggestions.
  • HEG HEG 1 min. p T T C T T T T T C T T T C p p T T C T T T T C T T T C p
  • oligonucleotides were incubated in human plasma for varying time periods. Circular oligonucleotide 7 and the precursor to this circle, linear oligonucleotide 2, were incubated at a 50 ⁇ M concentration in plasma at 37°C.
  • linear oligonucleotide 2 When incubated in human plasma the half-life of linear oligonucleotide 2 was 20 min. In contrast, circular oligonucleotide 7 underwent no measurable nuclease degradation during a 48 hr incubation.
  • the half-life of a circular oligonucleotide is greater than 48 hr in human plasma, i.e. more than 140 times longer than a linear oligonucleotide having an equivalent sequence.
  • oligonucleotides can preferentially bind to an RNA, rather than a DNA, target.
  • T target 5 ' -A A G A A T A G A A A G-3 '
  • dU target 5 ' - A A G A A U A G A A A G-3 ' .
  • a circular oligonucleotide having SEQ ID NO. : 14 was also prepared:
  • linear oligonucleotide complementary to the T and dU targets was also synthesized (i.e. the linear oligonucleotide, SEQ ID NO.: 13):
  • Tm melting temperatures
  • the linear oligonucleotide binds more strongly to the T target than to the dU target, by an amount which is significantly larger than experimental error limits. This difference in Tm values corresponds to a difference in free energy of binding of 1.7 kcal/mole.
  • the circular oligonucleotide binds more strongly to the U target. Therefore, the circular oligonucleotide can exhibit a preference for an RNA target relative to the corresponding DNA target.
  • the increase in binding strength for a circular oligonucleotide to the RNA target corresponds to a free energy difference of 0.8 kcal/mole which indicates that at 37 °C an RNA target would be preferred by about 3:1 over a corresponding DNA target.
  • Circular oligonucleotide 6 (Fig. 3) bound to a dA 12 target with 9 kcal/mole greater stability than did a linear dT 12 oligonucleotide (Example 2).
  • This increase in stability demonstrates that a circular-oligonucleotide: target complex is thermodynamically favored over a linear-oligonucleotide: target.
  • a circular oligonucleotide can actually
  • duplex DNA target sequences to form a complex with one strand of the duplex.
  • a DNA duplex target with a fluorescein group on one strand and a tetramethylrhodamine group on the other strand was prepared using published procedures (Cardullo et al. 1988 Proc. Natl. Acad. Sci. USA 85: 8790; Cooper et al. 1990 Biochemistry 29: 9261).
  • the structure of the duplex target (SEQ ID NO.: 15) was as follows:
  • the fluorescent substituents had no significant effect upon association kinetics. Moreover, the emission maxima of the fluoescein-dA 12 strand was 523 nm while the emission maxima of the rhodamine-dT 12 strand was 590 nm, allowing the association kinetics of the two strands could be separately monitored.
  • Strand displacement reactions were done at 10°C in a 1 cm fluorescence cuvette. Reaction conditions were 100 mM NaCl, 10 mM Mg Cl 2 and 10 mM Tris-HCl, pH 7.0 with a reaction volume of 3 ml. Labeled duplex was allowed to equilibrate for at least 1 hr at 10°C before addition of a 40-fold excess of linear or circular oligonucleotide (final concentration 0.01 ⁇ M).
  • a Spex Flurolog F 111A fluorescence instrument with 5 mm slit widths was used. An excitation wavelength of 450 nm and a monitored emission wavelength of 523 nm was used. The results were independent of both excitation and monitored emission wavelengths. Reactions were followed for at least 5 half-lives.
  • association rate constant of the two fluorescently-labeled strands was determined by mixing the strands under pseudo-first order conditions and monitoring the rate of decrease in fluorescein emission. At 10 °C the observed association constant was 3.2 X 10 6 M -1 sec -1 , which agrees well with published rates of association for DNA oligonucleotides (Nelson et al. 1982 Biochemistry 21: 5289; Turner et al. 1990 in Nucleic Acids (subvolume C), W. Saenger, Ed. Springer-Verlag, Berlin: 201-227).
  • Fig. 8 depicts a typical kinetic assay for the dissociation of duplex target by a 40-fold excess of unlabeled dA 12 (dotted line) or circular oligonucleotide 6 (solid line) at 10 °C.
  • duplex target dissociation by the circular oligonucleotide is
  • the first order rate constant for dissociation by the linear oligonucleotide is 2.0 X 10 -4 sec -1 whereas the first order rate constant for dissociation by the circular oligonucleotide is 2.3 X 10 -2 sec -1 , almost two orders of magnitude faster. This difference is even more apparent when the half-lives for the target duplex in the presence of linear vs circular oligonucleotides are calculated. At 10 °C, the duplex has a half-life for dissociation of 58 min in the
  • the rate of reaction between the circular oligonucleotide and duplex is dependent on the concentration of added circular oligonucleotide at low concentrations, and shows Michaelis-Menten type saturation behavior at higher concentrations (Fig. 9).
  • the dissociation rate of labeled duplex at 10°C can be derived from the duplex association rate constant and ⁇ G° 10 values. This rate constant, 8.5 ⁇ 10 -10 sec -1 , is consistent with rates derived from predicted
  • thermodynamic parameters for a duplex complex (Breslauer et al. 1986 Proc. Natl. Acad. Sci. USA 83: 3746) although this rate is significantly slower than the rate constant for strand displacement by a linear oligonucleotide.
  • An increase in duplex dissociation upon addition of a linear oligonucleotide has been noted in other cases (Chamberlin et al. 1965 J. Mol. Biol. 12: 410).
  • Comparison of the rate for the circular oligonucleotide-catalyzed reaction over that of the unassisted duplex dissociation reveals a rate enhancement of about 10 7 fold (Sigler et al. 1962 J. Mol. Biol. 5: 709) .
  • oligonucleotide] vs. 1/k obs is linear and yields a k cat of 0.024 ⁇ 0.005 sec -1 and a K M of 2.2 ⁇ 10 -7 M.
  • the k cat is 100-fold greater than the observed rate constant obtained for the reaction of the duplex with either dA 12 or dT 12 single strands.

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Abstract

Cette invention concerne des oligonucléotides circulaires à brin unique comprenant chacun un domaine (P) de liaison parallèle et un domaine (AP) de liaison antiparallèle séparés l'un de l'autre par des domaines à boucle. Chaque domaine P et AP présente une complémentarité suffisante pour se fixer sur un brin d'une cible définie d'acide nucléique dans laquelle le domaine P se fixe d'une manière parallèle sur la cible, et le domaine AP se fixe d'une manière antiparallèle sur la cible. En outre, les oligonucléotides circulaires à brin unique de cette invention peuvent se lier à des acides nucléiques cibles à brin unique et à des acides nucléiques à brins doubles. Cette invention concerne également des procédés d'utilisation de ces oligonucléotides ainsi que des compositions pharmaceutiques contenant ces derniers.
EP92912127A 1991-03-27 1992-03-26 Single-stranded circular oligonucleotides Withdrawn EP0579771A4 (en)

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US5683874A (en) * 1991-03-27 1997-11-04 Research Corporation Technologies, Inc. Single-stranded circular oligonucleotides capable of forming a triplex with a target sequence
FR2675803B1 (fr) * 1991-04-25 1996-09-06 Genset Sa Oligonucleotides fermes, antisens et sens et leurs applications.
IL107934A0 (en) * 1992-12-08 1994-04-12 Genta Inc Formation of triple helix complexes
US6096880A (en) * 1993-04-15 2000-08-01 University Of Rochester Circular DNA vectors for synthesis of RNA and DNA
US7135312B2 (en) 1993-04-15 2006-11-14 University Of Rochester Circular DNA vectors for synthesis of RNA and DNA
US6077668A (en) * 1993-04-15 2000-06-20 University Of Rochester Highly sensitive multimeric nucleic acid probes
US5714320A (en) * 1993-04-15 1998-02-03 University Of Rochester Rolling circle synthesis of oligonucleotides and amplification of select randomized circular oligonucleotides
US5473060A (en) * 1993-07-02 1995-12-05 Lynx Therapeutics, Inc. Oligonucleotide clamps having diagnostic applications
US5674683A (en) * 1995-03-21 1997-10-07 Research Corporation Technologies, Inc. Stem-loop and circular oligonucleotides and method of using
ES2109177B1 (es) * 1995-10-11 1998-07-16 Univ Barcelona Procedimiento general de preparacion de oligonucleotidos ciclicos e intermedios para el mismo.
US20050059016A1 (en) * 2002-11-05 2005-03-17 Ecker David J. Structural motifs and oligomeric compounds and their use in gene modulation
US7138384B1 (en) 1997-08-29 2006-11-21 The Regents Of The University Of California Modulators of DNA cytosine-5 methyltransferase and methods for use thereof
DK1034262T3 (da) 1997-11-18 2005-11-28 Pioneer Hi Bred Int Sammensætninger og fremgangsmåder til genetisk modifikation af planter
EP1032692A1 (fr) * 1997-11-18 2000-09-06 Pioneer Hi-Bred International, Inc. Manipulation ciblee sur des vegetaux de genes de resistance aux herbicides
US7102055B1 (en) 1997-11-18 2006-09-05 Pioneer Hi-Bred International, Inc. Compositions and methods for the targeted insertion of a nucleotide sequence of interest into the genome of a plant
EP1112378A1 (fr) * 1998-07-17 2001-07-04 GeneTag Technology, Inc. Procedes de detection et de mappage de genes, de mutations et de sequences de polynucleotides du type variant
US7560622B2 (en) 2000-10-06 2009-07-14 Pioneer Hi-Bred International, Inc. Methods and compositions relating to the generation of partially transgenic organisms
JP2005102502A (ja) * 2001-11-21 2005-04-21 Wakunaga Pharmaceut Co Ltd 一本鎖目的核酸断片の増幅方法
EP1986697B1 (fr) * 2006-02-17 2016-06-29 GE Healthcare Dharmacon, Inc. Compositions et procédés permettant l'inhibition de silençage de gènes par l'interférence arn
WO2008017473A2 (fr) 2006-08-08 2008-02-14 Gunther Hartmann Structure et utilisation d'oligonucléotides 5'-phosphate
JP5540312B2 (ja) * 2008-02-15 2014-07-02 独立行政法人理化学研究所 環状１本鎖核酸複合体およびその製造方法
JP5689413B2 (ja) 2008-05-21 2015-03-25 ライニッシュ フリードリッヒ−ウィルヘルムズ−ユニバーシタット ボン 平滑末端を有する５’三リン酸オリゴヌクレオチドおよびその使用
EP2508530A1 (fr) 2011-03-28 2012-10-10 Rheinische Friedrich-Wilhelms-Universität Bonn Purification d'oligonucléotides triphosphorylés au moyen d'étiquettes de capture
EP2712870A1 (fr) 2012-09-27 2014-04-02 Rheinische Friedrich-Wilhelms-Universität Bonn Nouveaux ligands de RIG-I et procédés pour les produire

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0128332A1 (fr) * 1983-05-05 1984-12-19 Enzo Biochem, Inc. Méthode d'essai employant des séquences de polynucléotides

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4766062A (en) * 1984-05-07 1988-08-23 Allied Corporation Displacement polynucleotide assay method and polynucleotide complex reagent therefor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0128332A1 (fr) * 1983-05-05 1984-12-19 Enzo Biochem, Inc. Méthode d'essai employant des séquences de polynucléotides

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, - 1987 pages 7150-7159, XP002027687 ERIE D. ET AL.,: "A dumbbell-shaped, double-hairpin structure of DNA: a thermodynamic investigation" *
BIOCHEMISTRY, vol. 28, 1989, pages 268-273, XP002027684 ERIE D. ET AL.,: "Melting behavior of a covalently closed, single-stranded, circular DNA" *
J. CHEM. SOC., CHEM. COMMUN.,, - 1 September 1991 pages 1161-1163, XP000647558 PRAKASH G. & KOOL E.: "Molecular recognition by circular oligonucleotides. Strong binding of single-stranded DNA and RNA" *
NUCL. ACID. RESEARCH, 1990, pages 3557-3564, XP002027686 XODO L. ET AL., : "Spectroscopic and calorimetric investigation on the DNA triplex formed by d(CTCTTCTTTCTTTTCTTTCTTCTC) and d(GAGAAGAAAGA) at acid pH" *
SCIENCE, vol. 245, - 1 September 1989 pages 967-971, XP002027685 GRIFFIN L. ET AL., : "Recognition of Thymine-Adenine base pairs by Guanine in a pyrimidine triple helix motif" *
See also references of WO9217484A1 *

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