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WO1997014708A1 - Oligonucleotides contenant des derives nucleosides a substitution thiol et procedes d'utilisation desdits oligonucleotides - Google Patents

Oligonucleotides contenant des derives nucleosides a substitution thiol et procedes d'utilisation desdits oligonucleotides Download PDF

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Publication number
WO1997014708A1
WO1997014708A1 PCT/US1996/004525 US9604525W WO9714708A1 WO 1997014708 A1 WO1997014708 A1 WO 1997014708A1 US 9604525 W US9604525 W US 9604525W WO 9714708 A1 WO9714708 A1 WO 9714708A1
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Prior art keywords
oligonucleotide
cross
linked
thiol
bridged
Prior art date
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PCT/US1996/004525
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English (en)
Inventor
Eric T. Kool
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Research Corporation Technologies, Inc.
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Publication of WO1997014708A1 publication Critical patent/WO1997014708A1/fr

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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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the present invention provides C-5 thiol-substituted nucleosides such as C-5 thiopropyne-substituted thymidine derivatives and oligonucleotides containing such derivatives.
  • the presence of the nucleoside derivatives in a linear oligonucleotide allows the formation of covalent cross-links between noncomplementary DNA domains.
  • Appropriate positioning of the thiol-substituted nucleoside derivatives in circular oligonucleotides permits disulfide links bridging the circle.
  • the resultant cross-linked linear and bridged circular oligonucleotides can bind nucleic acid targets with high affinity and
  • the subject oligonucleotides are useful for example as probes for target nucleic acids present in tissues or immobilized on membranes, and as
  • Ligands capable of sequence-specific binding to target nucleic acids have widespread diagnostic and therapeutic utility, for example as probes for target nucleic acids present in tissues or immobilized on membranes, and as regulators of
  • nucleic acid-binding ligands Recent advances in the design of such nucleic acid-binding ligands have resulted in novel structures that can bind to target nucleic acids with higher affinity than is possible with standard Watson-Crick base pairing. For example, one strategy for the binding of single-stranded DNA is the combined use of two
  • oligonucleotide binding domains that form a triple helical complex with a single-stranded target.
  • the binding domains may be present in two physically separate strands (Reynolds et al., 1994, Proc. Natl. Acad. Sci. USA 91 : 12433), or may be physically linked. Physical linking of two binding domains generally provides a significant advantage in binding affinity since the binding is less disfavored.
  • two binding domains are present in one double-length oligonucleotide and separated by a nucleotide or non-nucleotide loop.
  • the oligonucleotide can fold back around a single-stranded target to form a triplex. Giovannengeli et al.
  • Linking of the two binding domains at both ends can provide even greater binding advantages.
  • Circular oligonucleotides in which opposing
  • pyrimidine binding domains are bridged on both ends by nucleotide or nonnucleotide loops, bind to single-stranded DNA and RNA targets with very high affinity and very high sequence selectivity relative to simple Watson Crick complements.
  • An additional advantage of the cyclic structure is that circular oligonucleotides are quite resistant to degradation in human serum. Rumney et al. (1992) Angew. Chem. 104: 1686.
  • Stabilization of the triple helical complex is desirable, particularly for in vivo applications.
  • disulfide cross-links between the two noncomplementary oligonucleotide binding domains can strongly stabilize triple helical complexes formed with single-stranded target nucleic acids.
  • the present invention provides a thiol-carrying nucleoside that is not destabilizing to helices. It has further been discovered in accordance with the present invention that disulfide bonds can be used to cross-link two noncomplementary domains of nucleic acids. Linear and circular oligonucleotides in which noncomplementary domains are cross-linked provide ligands capable of binding to target nucleic acids with high affinity and sequence selectivity.
  • the present invention is directed to thiol-substituted nucleosides wherein the C-5 position of a pyrimidine base is modified to carry a thiolpropyne, thiolpropene, or thiolbutyne group.
  • the thiol-derivatized nucleoside is a C-5 thiopropyne-substituted thymidine analog including 5-(3-thiopropyn-1-yl)-2'-deoxyuridine.
  • the present invention also provides analogs of thiol-substituted nucleosides useful in the
  • the analog is 5-(3- Benzoylthiopropyn-1-yl)-2'-deoxyuridine 3', 5'-di-tert-Butyldimethylsilyl ether useful in the synthesis of the thiopropynyl deoxyuridine nucleoside of the present invention.
  • the analog is 5-(3-Benzoylthiopropyn-1-yl)-2'-deoxyuridine.
  • the analog is 5-(3-Benzoylthiopropyn-1-yl)-2'-deoxy-5'- 0-(4,4'-dimethoxytrityl)undine.
  • Another embodiment is directed to the analog 5-(3-Benzoylthiopropyn-1-yl)-2'deoxy-5'- 0-(4,4'-dimethoxy-trityl)undine 3'-0-(2-cyanoethyl N,N-diisopropylphosphoramidite).
  • the invention further provides linear oligonucleotides containing at least one C-5 thiol-substituted nucleoside derivative and circular
  • oligonucleotides containing at least two C-5 thiol-substituted nucleoside derivatives are oligonucleotides containing at least two C-5 thiol-substituted nucleoside derivatives.
  • Another aspect of the present invention is directed to cross-linked linear, cross-linked hairpin and bridged circular oligonucleotides containing at least two C-5 thiol-substituted nucleoside derivatives and at least one resultant disulfide cross-link between noncomplementary domains.
  • the present invention further provides a method of detecting a target nucleic acid which comprises contacting a sample containing a target nucleic acid with a cross-linked linear, cross-linked or hairpin or bridged circular oligonucleotide containing at least two C-5 thiol-substituted
  • nucleoside derivatives for a time and under conditions sufficient to form an oligonucleotide-target complex, and detecting the complex.
  • Another aspect of the present invention provides a method of regulating biosynthesis of a DNA, RNA or protein.
  • This method comprises contacting one of the subject cross-linked linear, cross-linked hairpin or bridged circular oligonucleotides of the present invention with a nucleic acid template for the DNA, RNA or protein under conditions sufficient to permit binding of the subject oligonucleotide to a target sequence within the template and detecting said binding.
  • kits for the synthesis of an oligonucleotide containing at least one C-5 thiol-substituted nucleoside derivative includes at least a first container containing a C-5 thiol-substituted nucleoside derivative such as a
  • Yet another aspect of the present invention provides a compartmentalized kit for the detection or regulation of a target nucleic acid comprising a first container containing a cross-linked linear, cross-linked hairpin or bridged circular oligonucleotide and a second container containing a reporter molecule for labeling the subject oligonucleotide.
  • Fig. 1 illustrates the structural formula of Triostin A, a disulfide cross-linked DNA-binding macrocycle. Also illustrated is a bridged circular oligonucleotide of the present invention.
  • Fig. 2 illustrates a disulfide link formed between two thiopropynyluracils spanning across a triple helix.
  • Fig. 3 schematically illustrates four binding strategies for binding single-stranded DNA by triplex formation. Increasing the number of links between the binding domains (as shown from left to right) results in increased binding affinity.
  • Fig. 4 depicts the effect of a central cross-link on triplex formation. Shown are thermal denaturation curves at pH 7.0 for a three-stranded triple helix 13 nucleotides in length and for the same sequence cross-linked in the +2 geometry.
  • Fig. 5 is a photograph of stained 201 denaturing PAGE gel showing intermediates and products of synthesis of a bridged circular oligonucleotide.
  • Lane 1 is a mixture of unmodified circular (upper band) and linear (lower band) 36mers for size
  • Lane 2 is crude starting 36mer containing two thiol groups.
  • Lane 3 is a crude product after oxidation in the presence of 13mer template strand.
  • Lane 4 is crude products after BrCN-mediated
  • Lane 5 is purified bridged circular (bicyclic) product.
  • Land 6 is treated bicyclic compound with dithiothreitol, showing mobility similar to that of unmodified cyclic 36mer.
  • Fig. 6 shows a comparison of binding behavior, as determined by thermal denaturation at pH 7.0, for three triple helical complexes having the same sequence but different linkage geometries as shown.
  • Fig. 7 depicts a comparison of sequence selectivities for three different types of DNA-binding oligonucleotides. Shown are the free energy
  • Fig. 8 outlines the synthesis of thiopropynyldeoxyuridine phosphoramidite from 5-iodo-deoxyuridine.
  • Fig. 9 schematically illustrates synthesis of a bridged circular oligonucleotide.
  • the present invention provides C-5 thiol-substituted nucleoside derivatives such as C-5 thiopropyne-, thiopropene- or thiobutyne-substituted nucleosides and oligonucleotides containing such derivatives.
  • the pyrimidine base of the subject C-5 thiol-substituted nucleoside derivatives comprises thymine, cytosine or uracil.
  • the subject nucleoside derivatives when incorporated into triplex-forming domains of linear, hairpin or circular oligonucleotides, allow the formation of disulfide cross-links between
  • the cross-linked (linear or hair pin) and bridged cyclic oligonucleotides are capable of binding to a target strand to form a triplex.
  • the subject nucleoside analogs are not destabilizing to a triple helix and in fact have surprisingly been found to provide increased stability to triplex formation.
  • sequence specific binding i.e. selective binding of the cross-linked oligonucleotides to a target can be achieved.
  • the C-5 thiol-substituted nucleoside derivative is a C-5 thiopropyne-substituted deoxyuridine having the structural formula:
  • the C-5 thiol- substituted nucleoside derivative is a C-5
  • the C-5 thiol-substituted nucleoside derivative is a C-5 thiobutyne-substituted deoxyuridine having the structural formula:
  • the C-5 thiol-substituted nucleoside derivative is a C-5
  • thiopropyne-substituted thymidine analog such as 5-(3-thiopropyn-1-yl)-2'-deoxyuridine.
  • the structure of 5-(3-thiopropyn-1-yl)-2'-deoxyuridine is:
  • the present invention further provides analogs of thiol-substituted nucleoside derivatives which are useful in making and using a preferred thiol-substituted nucleoside derivatives of the present invention.
  • X, Y and Z are independently H or a protecting group.
  • deoxyuridine have the structural formula:
  • X, Y and Z are independently H or a protecting group .
  • X, Y and Z are independently H or a protecting group.
  • the present invention further provides analogs (intermediates) of thiopropynyl deoxyuridine useful in making and using a preferred thiopropynyl deoxyuridine.
  • One preferred derivative has the structural formula:
  • X, Y and Z are independently H or a protecting group.
  • a protecting group is defined herein as any group capable of preventing the atom to which it is attached from participating in a reaction.
  • Protecting groups and methods for their introduction are well known to one of ordinary skill in the art and include, for example, benzoyl, dimethyoxy trityl (DMT), monomethoxytrityl (MMT) and fluorenylmethoxycarbonyl
  • alkylphosphonamidites and phosphoramidites including for example, ⁇ -cyanoethylphosphoramidites.
  • the C-5 thiol-substituted nucleosides of the present invention can be made by alkynyl or alkenyl derivation of undine or cytidine at the C-5 position.
  • the hydroxypropene, hydroxypropyne and hydroxybutyne intermediates can be synthesized by using methods well known in the art. Preferably, the hydroxypropene, hydroxypropyne and hydroxybutyne intermediates can be obtained through commercial sources.
  • the C-5 thiopropyne-substituted thymidine derivatives of the present invention can be made by alkynyl derivatization of undine at the C-5 position.
  • the hydroxypropyne intermediate can be synthesized from 5-lododeoxyuridine and hydroxypropyne with palladium catalysts by methods known in the art and described for example by Glick et al. (1992) J. Am. Chem. Soc. 114 : 5447.
  • a mesylate intermediate is derived from the hydroxypropyne intermediate by addition of methanesulfonyl chloride, and thiobenzoate is then used to displace the mesylate to yield 5'-(3-benzoylthiopropyn-1-yl)-2'-deoxyuridine. Deprotection by standard methods yields 5'-(3-thiopropyn-1-yl)-2'-deoxyuridine.
  • oligonucleotides can be made by any of a myriad of procedures known for making DNA or RNA
  • oligonucleotides include enzymatic synthesis and chemical synthesis.
  • inventions may comprise a number of different
  • a cross-linked linear oligonucleotide employing the thiol derivatized nucloesides of the present invention comprise two opposing non
  • Such linear oligonucleotides form a triplex with a target DNA or RNA.
  • a cross-linked linear oligonucleotide comprising a single strand of DNA or RNA having an intra-molecular (intra-strand) disulfide bond formed by appropriate placement of at least one pair of thiol-carrying nucleotides of the present invention forms a duplex of improved stability and selectivity with a target DNA or RNA.
  • a cross- linked linear oligonucleotide can encompass either a cross-linked linear duplex forming oligonucleotide
  • intra-strand or a cross-linked linear triplex forming oligonucleotide (inter-strand).
  • a cross-linked hairpin oligonucleotide comprises two opposing strands of noncomplementary DNA or RNA which are linked at one end by a nucleotide (or non-nucleotide) loop.
  • the cross-linked hairpin oligonucleotides of the present invention have at least one disulfide bond bridging the noncomplementary strands by appropriate placement of the thiol
  • the bridged circular oligonucleotides of the present invention comprise two opposing strands of noncomplementary DNA or RNA linked at both ends by a nucleotide (or non-nucleotide) loop. These bridged circular oligonucleotides have at least one disulfide bond bridging the noncomplementary strands of the circle by appropriate placement of the thiol
  • cross-linked hairpin oligonucleotides and the bridged circular oligonucleotides form triplexes with target DNA or RNA.
  • Enzymatic methods of DNA oligonucleotide synthesis frequently employ Klenow, T7, T4, Taq or E. coli DNA polymerases as described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY). Enzymatic methods of 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
  • oligonucleotides enzymatically requires a template nucleic acid which can either be synthesized
  • linear oligonucleotide which can be synthesized chemically.
  • linear oligonucleotides can be prepared by PCR techniques as described, for example, by Saiki et al., 1988, Science 239:487.
  • oligonucleotides is well known in the art and can be achieved by solution or solid phase techniques.
  • linear 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 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.
  • phosphite triester method produce ol igonucleotides having 175 or more nucleotides while the H-phosphonate method works well for oligonucleotides of less than 100 nucleotides. If modified bases are incorporated into the oligonucleotide, and particularly if modified phosphodiester linkages are used, then the synthetic procedures are altered as needed according to known procedures. In this regard, Uhlmann et al. (1990,
  • Chemical Reviews 90: 543-584 provide references and outline procedures for making oligonucleotides with modified bases and modified phosphodiester linkages.
  • 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
  • oligonucleotides may be subjected to DNA sequencing by any of the known procedures, including Maxam and
  • Sequences of short oligonucleotides can also be analyzed by laser desorption mass spectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am. Chem. Soc. 104:976; Viari, et al., 1987, Biomed.
  • DNA oligonucleotides are synthesized by automated methods using a DNA synthesizer and ⁇ -cyanoethyl phosphoramidite
  • the coupling procedure for the thiol- propene, thiol-propyne, or thiol-butyne derivative may be the same as used for unmodified bases.
  • deprotection of the protecting groups on the thiol-carrying nucleotides, along with the S-benzoyl group, should be rapid since the thiol-derivatized nucleoside is unstable to concentrated NH 4 OH and other strong reagents commonly used in deprotection such as NaOH.
  • deprotection can preferably be carried out by treatment with triethylamine while on a solid support followed by cleavage with aqueous NH 4 OH and
  • PAC protecting groups are also known in the art to be useful for rapid deprotection. All bases in the oligonucleotide chain whether tiol-derivatized or not should be rapidly deprotected.
  • one or more thiol-carrying nucleosides can be any suitable thiol-carrying nucleosides.
  • two thiols do not occur when they are in the same base step.
  • two thiols come in closest proximity (within about 2-3 ⁇ ) when one thiol is one (hereinafter "+1") or two
  • Intact incorporation and successful deprotection of the thiol-substituted nucleoside derivative into an oligonucleotide can be established by treating the oligonucleotide with a thiol reactive group such as N-ethylmaleimide in order to make a stable thiol adduct followed by chemical or enzymatic digestion into nucleosides.
  • a thiol reactive group such as N-ethylmaleimide
  • oligonucleotide can be digested with the enzymes snake venom phosphodiesterase or calf alkaline phosphatase as described in Evaluating and Isolating Synthetic Oligonucleotides; Applied Biosystems Inc., 1992, Appendix 1. Analysis of the products is then performed e.g. by HPLC analysis. The presence of the maleimide adduct of a C-5 thiol-substituted nucleoside derivative is indicative of successful incorporation and deprotection of the C5-thiol-substituted
  • nucleoside derivative After synthesis and deprotection of the oligonucleotides containing one or more C-5 thiol- substituted nucleoside derivatives, two
  • oligonucleotide strands are cross-linked.
  • cross linking is performed using crude oligomers after deprotection.
  • a purine complement may be used as a template to bring the two pyrimidine strands, and thus the two thiols in close proximity.
  • the pyrimidine and purine strands are mixed in an appropriate buffer, for example containing approximately 10 mM Mg 2+ and 100 mM Na + at a pH in the range of about 5.0 to 8.0 and incubated at about 25°C for a number of hours. A temperature of 23°C and an incubation period of 6-8 hours is especially preferred.
  • the solution is then incubated at 4°C for at least 8 hours.
  • Standard analytical denaturing gels can be used to determine extents of reaction.
  • Cross linked oligomers will have a slower migration pattern when compared to unliked oligomers.
  • Resultant cross-linked oligonucleotides have improved binding properties over single stranded oligonucleotides of the prior art. Similar to
  • the cross-linked oligonucleotides of the present invention are stabilizing when bound to their complements as part of a triplex forming complex.
  • the subject cross-linked oligonucleotides however,
  • cross-linked oligonucleotides of the present invention are stable and can be stored for long periods of time.
  • nucleotide loops at both ends of a pyrimidine binding domain and a disulfide bond bridging the pyrimidine binding domain allows
  • Standard analytical denaturing gels may be used to determine extents of cross-linking. Disulfide bond formation may also be monitored by treatment of a representative sample with a strong disulfide reducing reagent such as dithiothreitol (DTT). Resultant cross linked oligonucleotides, in their reduced dithiol form, have a slower gel migration pattern.
  • DTT dithiothreitol
  • DTT treatment subjected to DTT treatment may also be performed.
  • Binding affinities of such oligonucleotides are considerably lower than for cross-linked oligomers.
  • an oligonucleotide is constructed having strategically placed thiol-substituted
  • the oligonucleotide has the same sequence as the target nucleic acid; this is the end-joining oligonucleotide, or adaptor.
  • 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. It is preferred that the ends of the precircle fall within a binding domain, not within a loop.
  • 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-HCl 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 hours of incubation at 4-37°C. Other divalent metals, such as Cu, Pb, Ca and Mg, can also be used.
  • Both the intramolecular disulfide formation and BrCN cyclization reaction may be carried out with crude oligonucleotides. Purification can be performed after both bonds are formed.
  • RNA circularization incorporates the appropriate nucleotide sequences, preferably in a loop domain, into an RNA
  • Enzymatic circle closure is also possible using DNA ligase or RNA ligase under conditions appropriate for these enzymes.
  • Circular oligonucleotides can be separated from the end-joining oligonucleotide by denaturing gel electrophoresis or melting followed by gel
  • the recovered circular oligonucleotide can be further purified by standard techniques as needed for its use in the methods of the present invention.
  • the end-joining oligonucleotide may be attached to a solid support and recovered by
  • the bridged circular oligonucleotides of the present invention possesses excellent binding
  • oligonucleotides of the prior art bind complementary targets with association constants which are several orders of magnitude higher than the standard Watson-Crick complements, the addition of a disulfide link as provided by the present invention increases binding affinity even further.
  • AP Watson-Crick anti-parallel
  • the schematic illustration set forth below shows a representative 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 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 stretches of nucleotide sequence whose lengths are sufficient to permit binding to multiple targets.
  • a cross-linked or bridged circular oligonucleotide has
  • a stretch of nucleotide sequence separating one pair of corresponding AP and P binding domains can constitute an AP or P domain for binding to another target.
  • the stretch of nucleotide sequence is a loop domain between the ends of the binding domain and serves to circularize the oligonucleotide.
  • a cross-linked or bridged circular oligonucleotide of the present invention includes, e.g., two pairs of corresponding binding domains, these pairs of corresponding binding domains can also bind separate target sites.
  • a cross-linked or bridged circular oligonucleotide has multiple AP and P domains
  • the corresponding targets need not be linked on one nucleic acid strand.
  • oligonucleotide when bound to a given target can be an AP or P domain for binding to a second target when the oligonucleotide releases from the first target.
  • the nucleotide 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. For example, 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
  • the nucleotide sequence of the target DNA or RNA can be known in full or in part.
  • the sequence of the P and AP domains are designed with the
  • the target sequence can be represented by a consensus sequence or be only partially known.
  • oligonucleotides 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. Likewise, if a portion of a target sequence is known, one skilled in the art can refer to the base pairing rules provided hereinbelow to design cross-linked and bridged
  • circular ligands which bind to that target with higher affinity than a linear oligonucleotide that has a sequence corresponding to that of the cross-linked or bridged circular oligonucleotide.
  • the present invention is also directed to cross-linked and bridged circular oligonucleotides having P and AP domains which are sufficiently
  • 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 absorption upon binding or melting.
  • 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.
  • these rules are satisfied to the extent needed to achieve binding of a cross-linked or bridged circular oligonucleotide to its nucleic acid target, i.e. the degree of complementarity need not be 100% so long as binding can be detected.
  • the general rules for determining the sequence of the P domain are thus:
  • 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
  • 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
  • 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 cross-linked or bridged circular oligonucleotide and its target.
  • 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
  • AP 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
  • 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
  • the P, AP and loop domains are not complementary to each other.
  • Table 1 summarizes the nucleotides that can form anti-parallel base pairs or parallel base pairs with a defined 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
  • oligonucleotide and their respective target molecules 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 sometimes be conveniently described by the
  • 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
  • the degree of complementary between the P domain and the target and the AP domain and the target need not be the same.
  • the degree of 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.
  • 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 cross-linked and bridged circular oligonucleotides and their respective targets is dependent upon the conditions under which that binding occurs. It is well known that binding, i.e. hybridization, between nucleic acid strands depends on factors besides the degree of mismatch between two sequences. Such factors include the GC constent of the region, temperature, ionic strength, the presence of formamide and types of counter ions present. The effect that these
  • oligonucleotide is made for use in vivo, no formamide will be present and the ionic strength, types of counter ions, and temperature correspond to
  • Binding conditions can be manipulated in vitro to optimize the utility of the present oligonucleotides. A thorough treatment of the qualitative and quantitative considerations involved in establishing binding conditions that allow one skilled in the art to design appropriate
  • oligonucleotides for use under the desired conditions is provided by Beltz et al., 1983, Methods Enzymol,
  • binding or “stable 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
  • 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
  • Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures.
  • a method which is widely used because it is so simple and reliable, involves observing a change in light absorption of a solution containing an oligonucleotide and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the
  • oligonucleotide has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide and target
  • T m melting temperature
  • Cross-linked and bridged 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.
  • the P or HAP domain can bind to the target nucleotides by forming non-Watson-Crick hydrogen bonds (Hoogstein) (Table 1).
  • Hoogstein non-Watson-Crick hydrogen bonds
  • the specific thiol-carrying nucleosides of the present invention make possible the formation of stable cross-links in triple helical DNAs .
  • the bridging of two triplex-forming DNA strands in the middle of the helix greatly improves binding properties of the oligonucleotides to the target.
  • Standard nucleotide loops which link triplex-forming domains are optimally 4 to 5 nucleotides in length. Prakash et al., (1991) J. Am. Chem. Soc, 114:3523- 3528.
  • the presence of at least one single cross-link in the subject oligonucleotides provdes binding affinites at least as high as oligonucleotides having a nucleotide loop.
  • a bridged circular ligand binds its complement with an estimated free energy of -25 kcal/mol (37°C), for a 14 kcal advantage over Watson-Crick binding.
  • the bridged circular ligand has an even more favorable free energy of complexation of -40 kcal/mol.
  • the bridged circular oligonucleotides of the present invention have the highest binding advantage over standard Watson-Crick binding at conditions near physiological ionic strength. Since noncovalent bonds are formed in both circular and bridged circular oligonucleotides, the increased affinity associated with bridged circular
  • oligonucleotides is the result of greater
  • 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 nucleotides. The most preferred lengths are 6 to 36 nucleotides.
  • the binding domains are separated by loop domains which can independently have from about 2 to about 2000 nucleotides and can themselves
  • 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 bridged circular oligonucleotides less expensive to produce. More significantly, bridged circular oligonucleotides
  • oligonucleotides with non-nucleotide loops are more resistant to nucleases and therefore have a longer biological half-life than linear oligonucleotides. Furthermore, loops having no charge, or a positive charge, can be used to promote binding by eliminating negative charge repulsions between the loop and target. In addition, bridged 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
  • the length of these chains are equivalent to about 2 to about 2000 nucleotides, with preferred lengths equivalent to about 3 to about 8 nucleotides. The most preferred length for these chains is
  • 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 most preferred structural design requirement is provided in Rumney and Kool, 1995, J. Am. Chem.
  • Covalent bonds for example disulfide bonds, may comprise the loop domain.
  • cross-linked linear, cross-linked hairpin and bridged circular oligonucleotides of the present invention are composed of single-stranded DNA
  • RNA or a mixture thereof Cross-linked or bridged circular oligonucleotides comprising DNA and RNA are referred to herein as chimeric oligonucleotides. All possible chimeric oligonucleotides, for example, chimeric cross-linked and bridged circular
  • oligonucleotides containing a DNA binding domain and an RNA binding domain, or RNA binding domains and DNA loops are contemplated by the present invention.
  • the base composition of the nucleotides can vary and may include guanine (G), adenine (A), thymine (T),
  • C cytosine
  • U uracil
  • Ribose When possible, either Ribose, deoxyribose, 2'-O-methylribose sugars, 2'-O-allyln-ribose and 2'-fluoro-2'-deoxy ribose may be used with these analogs.
  • Ribose, deoxyribose, 2'-O-methylribose sugars, 2'-O-allyln-ribose and 2'-fluoro-2'-deoxy ribose may be used with these analogs.
  • Nucleotide bases in an ⁇ -anomeric conformation can also be used in the cross linked linear, cross-linked hair pin or bridged circular oligonucleotides of the present invention.
  • Preferred nucleotide analogs are unmodified G, A, T, C and U nucleotides; pyrimidine analogs with lower alkyl, lower alkoxy, lower alkynyl, lower alkenyl, 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-methyluricil, diaminopurine, and nucleotides with a 2'-O-methylribose, 2'-fluorodeoxyribose or 2'-aminodeoxyribose moiety in place of ribose or deoxyribose.
  • the oligonucleotide circle comprises RNA in which some of the pyrimidines are C-5 methylated and some of the ribose moieties are 2'-O-methylribose.
  • 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.
  • composition of the binding domains may be designed to optimize binding to a particular target species. Optimization is
  • oligonucleotides For example, for in vivo use or diagnostic applications involving biological fluids, resistance to nucleases is a critical consideration. In other diagnostic applications, binding affinity can be optimized with less consideration of nuclease resistance. For example, for binding to a single-stranded DNA target in applications involving
  • a preferred cross-linked or bridged circular oligonucleotide contains a DNA P domain and a DNA AP domain.
  • the preferred cross-linked or bridged circular oligonucleotide For binding to a single-stranded RNA target, the preferred cross-linked or bridged circular oligonucleotide contain RNA in both P and AP binding domains. In both cases, affinity can be increased by incorporating methylated pyrimidine bases into the binding domains.
  • pyrimidine rich binding domains composed of RNA or 2'- O-methyl RNA are preferred. Affinity can be increased by incorporating methylated pyrimidine bases into the binding domains.
  • binding domains composed of RNA are preferred in embodiments in which susceptibility to nucleases in minimal. Again C-5 methylation of pyrimidine bases is preferred.
  • cross-linked or bridged circular oligonucleotides can be designed to selectively bind DNA versus RNA targets.
  • a cross-liiked or bridged circular oligonucleotide with DNA P and AP domains hybridizes to a complementary single-stranded DNA target with significantly higher affinity than to an RNA target having the same affinity.
  • cross-linked or bridged circular oligonucleotides composed of RNA P and AP domains generally exhibit a small preference for binding RNA targets.
  • the present invention provides several methods of preparing bridged 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.
  • 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 invention, including chemical methods employing, for example, known coupling agents like BrCN, N-cyanoimidazole ZnCl 2 , 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide and other
  • the ends of a precircle can be joined by condensing a 5' phosphate and a 3' hydroxy, or a 5' hydroxy and a 3' phosphate.
  • the cyclization reaction is performed after the cross linking reaction since free thiols are unstable in the presence of chemical coupling reagents.
  • 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
  • oligonucleotides with poly-L-lysine can aid
  • 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.
  • protein carriers are useful when associated with or linked to the cross-linked or bridged circular oligonucleotides of this invention. Accordingly, the present invention contemplates derivatization of the subject cross-linked or bridged circular oligonucleotides of this invention. Accordingly, the present invention contemplates derivatization of the subject cross-linked or bridged circular oligonucleotides of this invention. Accordingly, the present invention contemplates derivatization of the subject cross-linked or bridged circular oligonucleotides of this invention. Accordingly, the present invention contemplates derivatization of the subject cross-linked or bridged circular oligonucleotides of this invention. Accordingly, the present invention contemplates derivatization of the subject cross-linked or bridged circular oligonucleotides of this invention. Accordingly, the present invention contemplates derivatization of the subject cross-linked or bridged circular oligonucleotides of this invention. Accordingly, the present invention contemplates derivatization of
  • 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, diterpenes and steroids.
  • 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 For example, viral DNA or RNA can be targeted for destruction without harming cellular nucleic acids by administering a cross-linked or bridged circular oligonucleotide (complementary to the targeted nucleic acid) which is linked to an agent that, upon binding, can cut or render the viral DNA or RNA inactive.
  • 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, chlororambucil, nitrogen mustards, psoralens,
  • 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 cross linked or bridged circular
  • oligonucleotides are provided.
  • derivatization of selected nucleotides permits incorporation of the group into selected domains of the cross linked or bridged circular oligonucleotide.
  • certain groups into a loop where that group will not interfere with binding, or into an AP or P domain to facilitate cleavage or modification of the target nucleic acid.
  • modification in the phosphodiester backbone of the cross-linked or bridged circular oligonucleotides is also contemplated. Such modifications can aid uptake of the oligonucleotide by cells or can extend the biological half-life of such nucleotides.
  • 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 can be replaced.
  • 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, phosphoramidates,
  • phosphotriesters and phosphoryl-boronates (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. Method for synthesis of oligonucleotides with modified
  • Bridged circular oligonucleotides with non-nucleotide loops can be prepared by any known
  • 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 invention.
  • 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.
  • 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). Also, different nucleotide sugars, for example 2'-O-methylribose can be incorporated into the oligonucleotides of this invention.
  • RNA 2'-O-methyloligoribonucleotides and DNA oligonucleotides differ only slightly.
  • RNA 2'-O-methyl oligonucleotides 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).
  • the present invention contemplates a variety of utilities for the subject cross linked and bridged circular oligonucleotides which are made possible by their selective and stable binding properties with complementary targets.
  • Some utilities include, but are not limited to: use of cross linked and bridged circular oligonucleotides of defined sequence, bound to a solid support, for affinity isolation of complementary nucleic acids; use of the subject oligonucleotides to provide sequence specific stop signals during polymerase chain reaction (PCR);
  • oligonucleotides covalent attachment of a drug, drug analog or other therapeutic agent to the subject oligonucleotides to allow cell type specific drug delivery; labeling the subject oligonucleotides with a detectable reporter group for localizing, quantitating or identifying complementary target nucleic acids; and binding cross linked and bridged circular oligonucleotides to a cellular or viral nucleic acid template and regulating biosynthesis directed by that template.
  • the subject cross-linked or bridged circular oligonucleotides can be attached to a solid support such as silica, cellulose, nylon, polystyrene,
  • Tentagel® polyethylene glycol, polyacrylamide, agarose and other natural or synthetic materials that are used to make beads, filters, and column
  • a cross linked or bridged circular oligonucleotide attached to a solid support can then be used to isolate a
  • incorporating a subject oligonucleotide solid support into a column for chromatographic procedures.
  • Other isolation methods can be accomplished without incorporation of the subject oligonucleotide: solid support into a column, e.g. by utilization of
  • Cross-linked or bridged circular oligonucleotide solid supports can be used, for example, to isolate poly(A) + mRNA from total cellular or viral RNA by making a subject
  • cross-linked or bridged circular oligonucleotides of the present invention may also be used to isolate single stranded or double stranded plasmid DNA from mixtures.
  • Cross-linked linear, hairpin or bridged circular oligonucleotide with P and AP domain poly(dT) or poly(U) sequences.
  • cross-linked or bridged circular oligonucleotides of the present invention may also be used to isolate single stranded or double stranded plasmid DNA from mixtures.
  • oligonucleotides are ideally suited to applications of this type because they are nuclease resistant and bind target nucleic acids so strongly.
  • PCR technology provides methods of synthesizing a double-stranded DNA fragment encoded in a nucleic acid template between two known nucleic acid sequences which are employed as primer binding sites. In some instances it is desirable to produce a single-stranded DNA fragment before or after having made some of the double-stranded fragment, or to selectively prevent
  • amplification of a particular species can be done by, for example, binding a cross-linked or bridged circular oligonucleotide of the present invention to one of the primer binding sites or to a site lying between the primer binding sites.
  • the present invention also contemplates use of the subject cross-linked or bridged circular oligonucleotides for targeting drugs to specific cell types.
  • Such targeting can allow selective destruction or enhancement of particular cell types, e.g.
  • mRNA for cell type specific drug delivery by the subject oligonucleotides linked to drugs or drug analogs.
  • Cells with high concentrations of target mRNA are targeted for drug delivery by administering to the cell a cross linked or bridged circular oligonucleotide with a covalently linked drug that is complementary to the target mRNA.
  • the present invention also contemplates labeling the subject cross-linked or bridged circular oligonucleotides for use as probes to detect a target nucleic acid. Labeled cross-linked or bridged
  • circular oligonucleotide probes have utility in diagnostic and analytical hybridization procedures for localizing, quantitating or detecting a target nucleic acid in tissues, chromosomes or in mixture of nucleic acids.
  • Labeling of a cross-linked or bridged oligonucleotide can be accomplished by incorporating nucleotides linked to a reporter group into the subject circular oligonucleotides.
  • a reporter group as defined herein, is a molecule or group which, by its chemical nature, provides an identifiable signal allowing detection of the circular oligonucleotide. Detection can be either qualitative or quantitative.
  • the present invention contemplates using any commonly used reporter molecule including radionuclides, enzymes, biotins, psoralens, fluorophores, chelated heavy metals, digoxigenin and luciferin.
  • the most commonly used reporter groups 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 ⁇ -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. For example, 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. Fluorophores may be detected, for example by microscopy or digital imaging. Similarly, methods for detecting
  • radionuclides are well-known in the art.
  • the probes so generated have utility in the detection of a specific DNA or RNA target in, for example, Southern analysis, Norther 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
  • the present cross-linked or bridged circular oligonucleotides can be used in conjunction with any known detection or diagnostic procedure which is based upon hybridization of a probe to a target nucleic acid. Moreover, the present cross-linked or bridged 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
  • 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 linear or hair pin C-5 thiol carrying oligonucleotide, at least one second container providing reagents for cross-linking the oligonucleotide, at least one third container for labeling the cross linked oligonucleotide with a reporter molecule and at least one fourth container for isolating the cross linked linear or hair pin oligonucleotide.
  • the kit contains at least one first container providing reagents for making a precircle precursor for a bridged circular oligonucleotide, at least one second container providing reagents for labeling the precircle with a reporter molecule, at least one third container providing reagents for circularizing the precircle, at least one fourth container for cross-linking the circular oligomer and at least one fifth container providing reagents for isolating the labeled bridged circular oligonucleotide.
  • the present invention provides a kit for isolation of a template nucleic acid.
  • a kit has at least one first container providing a cross-linked or bridged circular oligonucleotide which is complementary to a target contained within the template.
  • the template nucleic acid can be cellular and/or viral poly(A) + and the target can be the poly(A) + tail.
  • cross-linked or bridged 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).
  • kit for the detection of any target nucleic acid which contains a cross-linked or bridged circular oligonucleotide of the present invention linked to a reporter group.
  • 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
  • kits disclosed herein can include any elements recognized or conventionally used by the skilled artisan for constructing, purifying and using cross-linked and/or bridged circular oligonucleotides.
  • 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 cross-linked or bridged 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 for binding to the template, from moving along the
  • RNA when the template is RNA, regulation can be accomplished by allowing selective degradation of the template.
  • RNA for example, RNA
  • templates bound by the subject cross-linked or bridged circular oligonucleotides are susceptible to
  • 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
  • RNA transcription RNA transcription, RNA splicing, RNA polyadenylation, RNA translocation and protein
  • regulating biosynthesis includes inhibiting, stopping, increasing,
  • Regulation may be direct or indirect, i.e. biosynthesis of a DNA, RNA or protein may be regulated directly by binding to a second template encoding a protein that plays a role in regulating the biosynthesis of the first DNA, RNA or protein.
  • 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 cross-linked or bridged circular primer.
  • oligonucleotide to the primer binding site, and thereby blocking access to that site.
  • inhibition of D ⁇ A replication can occur by binding a cross-linked or branched 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 site.
  • RNA polymerase recognizes and binds to specific start sequences, or promoters, on a D ⁇ A template. Binding of RNA polymerase opens the D ⁇ A template.
  • transcriptional regulatory elements include enhancer sequences, upstream
  • Oligonucleotide binding to these sites can block R ⁇ A polymerase and transcription factors from gaining access to the template and thereby regulating, e.g., increasing or decreasing, the production of R ⁇ A, especially mR ⁇ A and tR ⁇ A.
  • the subject oligonucleotides can be targeted to the coding region or 3'-untranslated region of the DNA template to cause premature
  • 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 cross-linked or bridged circular oligonucleotide to a negative transcriptional
  • Negative transcriptional regulatory elements include repressor sites or operator sites, wherein a repressor protein binds and blocks transcription. Oligonucleotides 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 maturation processes before being translocated into the cytoplasm for protein translation.
  • 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.
  • a specific pre-mRNA template can be inhibited by using the subject cross-linked or bridged 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 cross-linked or bridged circular oligonucleotides of this invention.
  • oligonucleotides having a variety of sequences that, taken together, are complementary to the desired group of splice junction and intron branch point sequences.
  • 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 a 3' end of the pre-mRNA. Hence, any of these steps can be inhibited by binding the subject oligonucleotides to the
  • RNA translocation from the nucleus to the cytoplasm of eukaryotic cells appears to require a poly(A) tail.
  • a cross-linked linear, cross- linked hair pin or bridged 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 thiol carrying thymine residues, and preferably about 20 residues.
  • oligonucleotide preferably has 6 to about 12 thymine residues.
  • Protein biosynthesis begins with the binding of ribosomes to an mRNA template, followed by
  • biosynthesis, or translation can thus be blocked or inhibited by blocking access to the template using the subject cross linked and bridged circular
  • oligonucleotides to bind to targets in the template mRNA.
  • 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 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.
  • the present cross-linked or bridged circular oligonucleotides which are prepared to bind to multiple target sites, e.g. by having more than one P or AP domain, can also be more effective at regulating the biosynthesis of a DNA, RNA or protein than oligonucleotides which can bind only one target site.
  • the binding of two sites within a gene can provide greater inhibition than achieved with single-site binding (Lisziewicz et al., 1992, Proc. Natl. Acad. Sci. USA 8911209; Maher et al., 1987, J. Arch. Biochem. Biophys. 253: 214-220; Tannock, I. F. in "The Basic Science of Oncology" 2nd ed.; Tannock, I.
  • the present methods of regulating the biosynthesis of a DNA, RNA or protein can also include binding to more than one target within a template. Binding to more than one target within a template can be achieved by binding separate cross-linked or bridged circular oligonucleotides.
  • binding to more than one target within a template can be achieved by binding cross-linked or bridged circular oligonucleotides having multiple P or multiple AP domains.
  • Some types of genetic disorders that can be treated by the cross-linked linear, hairpin or bridged circular oligonucleotides of the present invention include Alzheimer's disease, beta-thalassemia, osteogenesis imperfecta, some types of arthritis, sickle cell anemia and others.
  • Many types of viral infections can be treated by utilizing the cross-linked or bridged circular oligonucleotides of the present invention, including infections caused by hepatitis, influenza, rhinovirus, HIV, herpes simplex, papilloma virus, cytomegalovirus, Epstein-Barr virus, adenovirus, vesicular stomatitus virus, rotavirus and respiratory syncytial virus among others.
  • animal and plant viral infections may also be treated by administering the subject oligonucleotides.
  • a further aspect of this invention provides pharmaceutical compositions containing the subject cross-linked linear, hairpin or bridged circular oligonucleotides with a pharmaceutically acceptable carrier.
  • the subject 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 01 ⁇ 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
  • Supplementary active ingredients can also be provided.
  • compositions incorporated into the compositions.
  • the subject oligonucleotides may be administered topically or parenterally by, for
  • osmotic pump intravenous, intramuscular, intraperitoneal subcutaneous or interdermal route, or when suitably protected, the subject oligonucleotides may be orally administered.
  • the subject oligonucleotides may be orally administered.
  • 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 including liposomes modified with polyethylene glycol or admixed with cationic lipids for parenteral
  • substances into the liposome for example, antibodies reactive against membrane proteins found on specific target cells, can help target the oligonucleotides to specific cell types.
  • the present invention contemplates administering the subject cross-linked or bridged cyclic oligonucleotides with an osmotic pump providing continuous infusion of such oligonucleotides, for example, as described in Rataiczak et al. (1992) Proc. Natl. Acad. Sci. USA 89:11823-11827).
  • an osmotic pump providing continuous infusion of such oligonucleotides, for example, as described in Rataiczak et al. (1992) Proc. Natl. Acad. Sci. USA 89:11823-11827).
  • Such osmotic pumps are commercially available, e.g., from Alzet Inc. (Palo Alto, CA). Topical administration and parenteral administration in a cationic lipid carrier are preferred.
  • thiopropynyldeoxyuridine phosphoramidite The hydroxypropyne intermediate was synthesized from 5-iododeoxyuridine following the procedure of Glick et al. (1992) J. Am. Chem. Soc. 114:5447-5448.
  • Thiobenzoate was used to displace the mesylate derived from the hydroxypropyne in good yield. Subsequent deprotection and tritylation proceeded in good yields under standard conditions, followed by
  • 4,4'-Dimethoxytritylchloride was dried over P 2 O 5 in a vacuum desiccator for 24 h prior to use.
  • Other reagents, chemicals and solvents were used as obtained from Aldrich, Sigma, Lancaster, Fisher Scientific or J.T. Baker Inc. Reagents, chemicals and solid
  • the thiobenzoate silyl ether (740 mg., 1.17 mmol) was treated with 1M n-Bu 4 NF/2M pyridinium hydrogen fluoride in dry pyridine (2.8 mL) for 20 hours at 23oC. After removal of pyridine under reduced pressure, the crude residue was absorbed on
  • tritylated nucleoside was obtained as a white foam.
  • DNA oligonucleotides were synthesized by automated methods on a ABI 392 DNA synthesi zer using ⁇ -cyanoethyl phosphoramidite chemistry on 0.2 or 1.0 umole scales.
  • the synthetic thiopropynyl nucleoside phosphoramidite coupled as 0.11M solution in dry CH 3 CN with >97% efficiency.
  • Ac-dC-CE phosphoramidite and Bz-dC-lcaa-CPG were used instead of the standard Bz-dC-CE phosphoramidite and Bz-dC-lcaa-CPG. All unmodified oligonucleotides were cleaved and
  • Thiol-modified oligomers were first treated on the solid support with dry triethylamine for 2 hours at 23°C and then were cleaved from solid support and deprotected with the UltraFAST System of Reddy et al. (1994) Tetrahedron Lett., 35:4311, (AMA, 50:50, V/V mix. of 29.5% NH 4 OH and 40% MeNH 2 in water) containing 330 mM dithiothreitol (DTT) at 23°C for 90 minutes. These oligomers were isolated by dialysis (water, 4 ⁇ 2.0L, 12h) and lyophilization, quantitated by UV absorbance at 260 nm, and were submitted to disulfide cross-linking reactions without further purification.
  • the thiopropyne modified phosphoramidite was first introduced at single sites in five 13mer and 14mer pyrimidine oligodeoxynucleotides having the sequence 5'-CTTCTTTTTCTTC or 5'-dCTTCTTTTTTCTTC (where underlined bases are each modified separately) (see Table 2).
  • nucleoside appeared to be unstable to concentrated NH 4 OH at 55°C for 8 hours.
  • sequence 5'-dCTTCTTXTTTCTTC was treated with N-ethylmaleimide to make a stable thiol adduct, and the oligomer was then enzymatically digested to
  • maleimide adduct of thiopropynyldeoxyuridine also confirmed by coinjection with an authentic sample.
  • the relative peak areas were consistent with the expected 9:4:1 ratio of nucleosides.
  • Solutions for the thermal denaturation studies contained a one-to-one ratio of a given pyrimidine oligomer and complementary purine target oligomer (1.5 uM each). Also present were 100 mM NaCl and 10 mM MgCl 2 . Solutions were buffered with 10 mM Na•PIPES (1,4-piperazine-bis(ethanesulfonate), Sigma) at the pH values indicated. The buffer pH is that of a 1.4X stock solution at 25°C containing the buffer and salts. After the solutions were prepared they were heated to 90°C and allowed to cool slowly to room temperature prior to the melting experiments.
  • thermoprogrammer equipped with thermoprogrammer.
  • Uncertainty in T m is estimated at ⁇ 0.5°C based on repetitions of experiments. Free energy values were estimated by nonlinear least squares fitting of the denaturation data, using a two-state model with linear sloping baselines. 40 Precision in individual free energy measurements is estimated at ⁇ 5-10% based on repetitions of experiments. For complexes with higher T m values the free energy is likely to be less
  • the sequence of the tested strands was 5'-dCTTCTXTTTCTTC, where X is thiopropynyldeoxyuridine or unmodified thymidine. Thermal denaturation studies of these strands hybridized to the complement
  • Asterisks indicate positions of the thiol-substituted nucleoside derivative, 5'(3-thiopropyn-1-yl)-2'deoxyuridine.
  • the above-delineated oligonucleotides have sequence symmetry which allows them to bind a purine complement in 2:1 ratio; forming pyrimidine-purine-pyrimidine triple helices.
  • the complexes are either 13 or 14 nucleotides in length, and differ by the presence or absence of one T-A-T triad near the center.
  • Use of the purine complement as a template served to bring two pyrimidine strands, and thus two thiols, in close proximity. Since the position of the thiol modification is varied systematically in these five sequences, the series was utilized to investigate whether the relative positioning of the two thiols would affect either cross-linking efficiency or binding efficiency of the resulting linked compounds.
  • Cross-linking was carried out using crude oligomers just after deprotection. Attempts at gel purification of these oligomers as the free thiols gave compounds which subsequently could not be cross-linked, and so gel purification was avoided until after disulfides were formed.
  • the cross-linking reactions were monitored by analytical denaturing gel electrophoresis; a successful reaction would be expected to give a band with mobility approximately equal to that of an oligomer twice the starting length.
  • the reactions were carried out by mixing the pyrimidine and purine strands in 2;1 ratio (3 ⁇ M concentration for the pyrimidine strand) in a pH 5.0 buffer containing 10 mM Mg 2+ and 100 mM Na + and
  • the resulting mixture was kept exposed to air at 23 C for 6-8 hours and at 0-4 C overnight.
  • the solution was then dialyzed against water (4 ⁇ 2.0L) for 16 hours and lyophilized n a speed-vac.
  • the dried cross-linked oligomers were purified by gel electrophoresis on 20% polyacrylamide under denaturing conditions followed by crush-soak and dialysis method and were quantitated by UV absorbance at 260 n.
  • Molar extinction coefficients of the cross-linked oligomers were calculated as the sum of the molar extinction coefficients of the two corresponding uncross-linked strands. Yields of the purified oligomers were in the range of 5.0-9.0 nmol (33-60%).
  • Example 6 The three cross-linked products of Example 6 were then examined for their ability to bind the complementary target sequence (either
  • Results show that all three of the linked strands form strong, cooperative complexes with the complements. All three complexes are pH dependent, displaying tighter binding at pH 5.0 than at neutral pH, indicating formation of triple helical complexes. All three complexes also show only a single sharp melting transition having similar hyperchromicity to that of the unmodified triplexes ( Figure 4). The latter, by contrast, show two clear transitions at neutral pH as a result of initial loss of the
  • oligonucleotide was synthesized having the sequence 5'-dCTTCTTTTTTCTTCTTTTTCTTCTTTTTTCTTC, which contains two copies of a 14-base binding domain (the second sequence listed in Example 6 and Table 2) linked with a pentanucleotide loop (underlined).
  • oligonucleotide with a 3'-phosphate was synthesized and deprotected as described in Examples 3 and 4.
  • the sequence of the oligonucleotide was: * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
  • Astericks indicate positions of the thiol-substituted nucleoside derivative, 5-(3-thiopropyn-1-yl)-2'-deoxyuridine.
  • the complementary target sequence for the final product was 5'-dAAAGAGAGAAA.
  • the bridged circular oligonucleotide was examined for its ability to bind a complementary DNA strand.
  • a circular oligonucleotide having the same sequence as the bridged circular one but with no modified nucleotides (Table 3) was synthesized for comparison purposes.
  • a linear 13-base oligonucleotide which is complementary to the same target in simple Watson-Crick fashion was also synthesized. Binding was evaluated by thermal denaturation experiments in buffers containing 100 mM Na+ and 10mM Mg 2+ and as described in Example 5.
  • the three-stranded complex melts in non-cooperative fashion and at considerably lower temperatures than the two complexes involving cyclic ligands. In contrast to this, the two bimolecular complexes melt with sharp single transitions.
  • the binding data show (Table 3) that the bridged circular oligonucleotide binds its complement with extremely high affinity. At neutral pH it binds the complement with a T m of 64.3°C and a free energy estimated at -25kcal/mol. This is nearly 10°C and 8 kcal/mol more favorable than binding by the unmodified circular oligomer, and it is 20°C and 15 kcal/mol more favorable than binding by a standard Watson-Crick complement. At pH 5.0 the affinity of the bridged circular ligand increases further, with a T m rising to 83.3°C.
  • Sequence selectivity was defined as the difference in free energy of binding the correctly matched target versus that for the mismatched targets, as determined by thermal denaturation experiments.
  • the selectivities of the bridged circular oligomer an unmodified circle having the same sequence (see Table 3 for sequences), and a Watson-Crick complement were compared both at pH 5.0 and pH 7.0.
  • Figure 7 compares the selectivities of these three oligonucleotide ligands at neutral pH. Results show that selectivity of the unmodified circle is considerably higher than that of the Watson-Crick complement, as has been previously observed. Kool et al., 1991, J. Am. Chem. Soc. 113:6265-6266; Wang et al., 1995, Biochemistry 34:9774-9784. Interestingly, the data show that the bridged circular ligand has even higher selectivity than the unmodified circular compound. At pH 7.0 the selectivity of the linear compliment against these single mismatches is 4.9-5.6 kcal/mol. The circular compound has a selectivity of
  • Solutions for thermal denaturation studies contained a one-to-one ratio of 34-nucleotide circular pyrimidine oligomer and complementary purine target oligomer (1.5 ⁇ M each). Also present were 100mM NaCl and 10 mM MgCl 2 . Solutions were buffered with 10 mM Na•PIPES (1,4-piperazine-bis(ethanesulfonate), Sigma) at the pH values indicated. The buffer pH is that of a 1.4X stock solution at 25°C containing the buffer and salts. After the solutions were prepared they were heated to 90°C and allowed to cool slowly to room temperature prior to the melting experiments.
  • thermoprogammer spectrophotometer equipped with thermoprogammer .

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Abstract

Nucléosides à substitution thiol C-5 tels que dérivés de thymidine à substitution thiopropyne C-5, et oligonucléotides contenant lesdits dérivés. La présence des dérivés nucléosides dans un oligonucléotide linéaire permet la formation de liaisons croisées covalentes entre des domaines d'ADN non complémentaires.
PCT/US1996/004525 1995-10-04 1996-03-29 Oligonucleotides contenant des derives nucleosides a substitution thiol et procedes d'utilisation desdits oligonucleotides WO1997014708A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999036429A2 (fr) * 1998-01-16 1999-07-22 The Perkin-Elmer Corporation Oligomeres nucleobases
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J.SAGI ET AL.: "Base-Modified Oligodeoxynucleotides. I. Effect of 5-Alkyl, 5-(1-Alkenyl) and 5-(1-Alkynyl) Substitution of the Pyrimidines on Duplex Stability and Hydrophobicity.", TETRAHEDRON LETTERS, vol. 34, no. 13, 26 March 1993 (1993-03-26), OXFORD GB, pages 2191 - 2194, XP002005977 *
J.T.GOODWIN ET AL.: "Incorporation of Alkylthiol Chains at C-5 of Deoxyuridine.", TETRAHEDRON LETTERS, vol. 34, no. 35, 27 August 1993 (1993-08-27), OXFORD GB, pages 5549 - 5552, XP002005978 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999036429A2 (fr) * 1998-01-16 1999-07-22 The Perkin-Elmer Corporation Oligomeres nucleobases
WO1999036429A3 (fr) * 1998-01-16 1999-11-25 Perkin Elmer Corp Oligomeres nucleobases
US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
US12269839B2 (en) 2017-06-30 2025-04-08 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use

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