WO1997006252A1

WO1997006252A1 - Synthetic oligonucleotides with enhanced ribonuclease activity

Info

Publication number: WO1997006252A1
Application number: PCT/US1996/011420
Authority: WO
Inventors: John Goodchild; Steven M. Nesbitt
Original assignee: Hybridon, Inc.
Priority date: 1995-08-08
Filing date: 1996-07-09
Publication date: 1997-02-20
Also published as: AU6455096A

Abstract

Disclosed are catalytic synthetic oligonucleotides having enhanced ability to site-specifically endonucleolytically cleave a sequence of 3' to 5' linked ribonucleotides. These synthetic catalytic oligonucleotides include at least one 5-propynyl substituted pyrimidine nucleotide at a flanking region. Also disclosed are methods of preparing and utilizing the synthetic catalytic oligonucleotides of the invention, and pharmaceutical formulations and kits containing such synthetic catalytic oligonucleotides.

Description

SYNTHETIC OLIGONUCLEOTIDES WITH ENHANCED RIBONUCLEASE ACTIVITY

BACKGROUND OF THE INVENTION

This invention relates to synthetic molecules with enhanced endonucleolytic activity useful in the site-specific cleavage of RNA. This invention also relates to the control of gene expression through the degradation of mRNA.

Some molecules containing nucleotides covalently linked through phosphate esters have the ability to endonucleolytically cleave at specific phosphodiester linkages in single- stranded RNA molecules to which they have hybridized. For example, ribozymes are catalytic RNA molecules with recognized structures which cleave mRNAs, RNA-containing substrates, and ribozymes, themselves, at particular sites.

Ribozyme selectivity or specificity, i.e., the ability to discriminate between all RNA molecules in a cell, depends on the number of base pairs formed between the ribozyme flanking sequences and its RNA substrate; to have the requisite selectivity or specificity, a ribozyme must form sufficient base pairs with the target substrate. Unfortunately, this requirement for selectivity limits the rate of cleavage that may be realized; increased base pairing has been shown to decrease the rate of cleavage. For example, the rate of cleavage of a particular HIV-l RNA by various hammerhead ribozymes was reported to be dependent on the length of the flanking sequence: shorter sequences were shown to resulting weaker binding between the ribozyme and the cleavage products, together with increased rate of cleavage (Goodchild and Kohli (1991) Arch. Biochem. Biophys. 284:386-391) .

Furthermore, although the endonucleolytic activity of ribozymes has been demonstrated in vitro, their use in vivo has been limited by their susceptibility to RNases. In addition, therapeutics such as ribozymes having greater than 30 or more nucleotides are expensive and difficult to produce in great quantities.

Thus, more recent development of ribozymes has focused on modifications which improve efficacy through (i) protection from nucleolytic cleavage (stability) ; and (ii) an increased rate of catalytic activity.

For example, in an effort to protect antisense oiigonucleotides from degradative influences in vivo, various structural modifications have been made to these molecules, including the replacement of phosphodiester linkages with non- phosphodiester linkages, the substitution of various sugar groups and bases, the addition of end-capping groups, and the substitution or replacement of existing structures with the self- hybridizing termini (reviewed in Goodchild (1990) Bioconjugate Chem. 1:165-187; Agrawal et al. (1992) Trends Biotechnol. 10 :152-158; WO 93/15194,* WO 94/10301; WO 94/12633) .

However, modifications which protect an RNA molecule from endonuclease digestion may also affect the catalytic activity of the ribozyme. For example, Perreault et al . (Nature (1990) 344:565-567) report that the replacement of ribonucleotides at various conserved positions within the ribozyme sequence with 2'- deoxynucleotides resulted in a 96% decrease of catalytic efficiency. Perreault et al . (Biochem. (1991) 30:4020-4025) and Dahm et al . (Biochem.

(1990) 72:819-23) disclose that the replacement of various 2'-hydroxyl groups with hydrogen atoms reduced the catalytic activity of hammerhead ribozymes. Olsen et al . (Biochem. (1991) 30:9735- 9741) report that replacing 2' -hydroxyl groups on all adenosine residues by either fluorine or hydrogen decreases the catalytic activity of a ribozyme. Odai et al . (FEBS Lett. (1990) 267:150- 152) report that replacing the exocyclic amino group of a conserved guanosine residue in the core region with hydrogen reduced catalytic activity. Ruffner et al. (Nucleic Acids Res. (1990) 18:6025-6029) and Buzayan et al . {Nucleic. Acids Res. (1990) 18:4447- 4451) disclose that replacing oxygen atoms by sulfur on various internucleotide phosphate residues reduces catalytic activity. Pieken et al. (Science (1991) 253:314-317) disclose that catalytic activity is reduced when various 2'- hydroxyl groups on adenosine residues are replaced with fluorine and when the 2'-hydroxyl groups on cytidine residues are replaced with amine groups. Paolella et al . (EMBO J. (1992) 11:1913-1919) have investigated which 2'-hydroxyl groups may or may not be alkylated without loss of catalytic activity.

Other groups have substituted nucleotides within the ribozyme, thereby forming nucleotide analogs. For example, Usman et al . (WO 93/15187) designed chimeric polymers or "nucleozymes" with ribozyme-like catalytic activity having ribonucleotides or nucleic acid analogs (with modified sugar, phosphate, or base) at catalytically critical sites and nucleic acid analogs or deoxyribo-nucleotides at non- catalytically critical sites. Ludwig et at (WO 94/13789) and McLaughlin et al. (WO 95/06764) disclose ribozymes or oiigonucleotides with RNA cleavage activity, respectively, having at least one 2' -substituted adenosine or guanosine derivative in a non-flanking region. Sproat et al. (U.S. Patent No. 5,334,711) disclose synthetic catalytic oligonucleotide structures with nucleotide analogs having a 2'-alkoxy substituent.

Modifications such as a reduction in the length of the helix II structure of the hammerhead ribozyme have been made in an effort to design a more stable molecule without reducing its catalytic activity (see, e.g., Goodchild et al . ( 1991 ) Arch. Biochem. Biophys. 284 : 386 - 391 ; Tuschl et al . ( 1993 ) Proc. Natl. Acad. Sci. (USA) 90 : 6991 - 6994 ; McCall et al . ( 1992 ) Proc. Natl. Acad. Sci. (USA) 89 : 5710 -

5714 ) .

Modifications in ribozyme structure have also included the substitution or replacement of various non-core portions of the molecule with non-nucleotidic molecules (see, e.g., U.S. Patent Application Ser. No. 08/315,287; U.S. Patent

Application Ser. No. 08/336,526; Benseler et al . (1993) J. Am. Chem. Soc. 115:8483-8484; Ma et al .

(1993) Biochem. 32:1751-1758; Ma et al. (1993) Nucleic Acids Res.21:2585-2589; and Thomson et al . (1993) Nucleic Acids Res. 21:5600-5603) . However, such substituted molecules heretofore have not demonstrated greatly improved catalytic properties.

The cleavage abilities of ribozymes and ribozyme analogs, especially those with small flanking regions, have been enhanced by introducing a facilitator oligonucleotide into the system which hybridizes adjacent one or both of the flanking regions of the ribozyme or ribozyme analog (WO 93/15194) .

Enhanced target affinity of antisense oiigonucleotides has been achieved with the use of certain pyrimidine nucleotides substituted at position 5 (see, e.g., Froehler et al . (1992) Tetrahedron Lett. 33:5307-5310; Sagi et al . (1993)

Tetrahedron Lett. 34:2191-2194; and Colocci et al . (1994) J. Am. Chem. Soc. 116:785-786) . Antisense oiigonucleotides containing such 5-propyne analogs can inhibit gene expression (see, e.g., Wagner et al. (1993) Science 260:1510-1513; Fenster et al . (1994) Biochem. 33:8391-8398) . Such 5-propyne analogs have heretofore not been incorporated into catalytically active oiigonucleotides.

Thus, ribozymes and other catalytic oiigonucleotides with improved nuclease resistance and increased specificity, coupled with enhanced ribonuclease activity, are desirable, and there continues to be a need for such catalytic molecules . A need also remains for improved methods of cleaving target RNA-containing molecules, and of controlling gene expression.

SUMMARY OF THE INVENTION

It is known that increased base pairing of a ribozyme to target molecule increases specificity but decrease the rate of target cleavage by the ribozyme. Surprisingly, it has been discovered that a single 5-propynyl substituted pyrimidine nucleotide can increase turnover of ribozymes having short flanking regions with few as five nucleotides. This is an unexpected result, as 5- propynyl substituted pyrimidine nucleotides are known to stabilize hybridization of an antisense oligonucleotide to its complementary target by increasing base stacking.

These findings have been exploited to provide the present invention, which includes compositions and methods for controlling gene expression, and methods for increasing ribozyme catalytic activity without reducing specificity or nuclease resistance.

In one aspect, the present invention provides a synthetic catalytic oligonucleotide having enhanced ribonuclease activity, i.e., an increased ability to endonucleolytically cleave single- stranded target RNA and RNA-containing substrates. Thus, synthetic oiigonucleotides according to the invention are useful as RNA-specific restriction endonucleases, and as such, in combination with RNA ligases, allow for the preparation of recombinant RNA molecules.

For purposes of the invention, the term "synthetic oligonucleotide" includes chemically synthesized polymers of 25 up to 54 and preferably from about 32 to about 34 ribonucleotide or ribonucleotide and deoxyribonucleotide monomers covalently linked by at least one, and preferably more than one, 5' to 3' internucleotide linkage.

As used herein, the terms "target RNA, " "substrate RNA," and "RNA-containing substrate" refer to an oligoribfpnucleotide, RNA/DNA hybrid, or RNA-containing molecule containing 3' to 5' covalently-linked ribonucleotides onto which the flanking regions of the synthetic oligonucleotide hybridize, and which the synthetic oligonucleotide recognizes and cleaves.

The synthetic catalytic oiigonucleotides of the invention comprise a 5' flanking region and a 3' flanking region, each flanking region having about five to fifteen nucleosides. At least a portion of the 3' flanking region is complementary to a first target region of a substrate RNA molecule, and at least a portion of the 5' flanking region is complementary to a second target region of the substrate RNA molecule. In some embodiments, the flanking regions of the synthetic catalytic oiigonucleotides of the invention have about four to fifteen nucleosides. Preferred embodiments have five to six nucleosides per flanking region.

At least one of the flanking regions of the catalytic oligonucleotide contains a pyrimidine nucleotide with a 5-propynyl substituent. In some embodiments, the 5-propynyl substituted pyrimidine nucleotide is a 2'-O-methyl ribonucleotide. Preferably, the substituted pyrimidine nucleotide is uracil, deoxy-uracil, cytidine, or deoxy¬ cytidine.

In one embodiment, the synthetic oligonucleotide contains the 5-propynyl substituted pyrimidine nucleotide in its 3' flanking region. In another embodiment, the synthetic oligonucleotide contains the 5-propynyl substituted pyrimidine nucleotide in its 5' flanking region. In yet another embodiment, the synthetic oligonucleotide contains 5-propynyl substituted pyrimidine nucleotides at both its 5' and 3' flanking regions. In some embodiments, the 5-propynyl substituted nucleotide is the terminal or penultimate nucleotide of either the 3' or 5' flanking region, of both flanking regions. In other embodiments, two or more 5-propynyl substituted pyrimidine nucleotides are located in either or both flanking regions. In yet other embodiments, all of the pyrimidine nucleotides in one or both flanking region(s) is (are) 5-propynyl substituted.

In one particular embodiment, the synthetic catalytic oligonucleotide contains, in addition to the 3' and 5' flanking regions, a nucleotidic stem-loop region, and first and second nucleotidic core regions forming a catalytic core. The nucleotidic stem-loop region has a 3' terminus and a 5' terminus and comprising a plurality of 3 ' to 5' covalently-linked, self-hybridizing nucleotides. The first and second nucleotidic core regions each comprises a plurality of 3' to 5' covalently-linked nucleotides, and each has a

3' terminus and a 5' terminus . The 3' terminus of the first nucleotidic core region is covalently linked to the 5' terminus of the stem-loop region, and the 5' terminus of the second nucleotidic core region is covalently linked to the 3' terminus of the stem-loop region. The catalytic core is flanked by the 3' and 5' flanking regions, the 3' and 5' flanking regions, the 3' terminus of the first flanking region being covalently linked to the 5' terminus of the first nucleotidic core region, and the 5' terminus of the second flanking region being covalently linked to the 3' terminus of the second nucleotidic core region.

In another embodiment, the synthetic catalytic oligonucleotide has a ribozyme-like structure, and contains, in addition to the 3' and 5' flanking regions, a helix II having a 3' terminus and comprising a stem region and a loop region. The stem region also has a 3' terminus and 5' terminus and includes a plurality of 3' to 5' covalently-linked, self-hybridized nucleotides. As used herein, the term "self-hybridizing" refers to nucleotides in the stem region of the helix II which are complementary to each other, and which form normal Watson-Crick base pairs. As used herein, the term "helix II" refers to the double- stranded, coiled helical structure in hammerhead ribozymes having at one end a single- stranded loop, as described by Haseloff et al . (Nature (1988) 334:585-591) . The stem region has two complementary nucleotidic strands which include at least one nucleotide on one stand and one nucleotide on the other strand which base pair together. The loop region of the helix II is covalently linked to the stem region at its 3' and 5' termini and comprises a plurality of 3 ' to 5' covalently-linked nucleotides.

Preferred embodiments of the invention include synthetic catalytic oiigonucleotides having at least one modified nucleotide, in addition to the 5-propynyl substituted pyrimidine nucleotide (s) . As used herein, the term "modified nucleotide" refers to a nucleotide which has a modified structure not usually found in nature.

Modifications include additions to, reductions in, or substitutions in any portion of the nucleotide include its sugar, base, or side groups.

In one embodiment, all of the pyrimidines in the 5' and 3' flanking regions are alkylatedon the 2' hydroxyl group. In other embodiments, the nucleosides in the first and second flanking regions are covalently linked with alkylphosphonate, phosphorothioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, acetamidate, or carboxymethyl ester internucleotide linkages, or a combination of such linkages.

In another aspect, the invention provides a pharmaceutical formulation including a synthetic catalytic oligonucleotide in a physiologically acceptable carrier. In some embodiments, the pharmaceutical formulation contains at least two oiigonucleotides of claim 1 having different 3' and 5' flanking regions, and thus are targeted to different sequences of the target molecule.

The invention also provides kits containing at least one synthetic catalytic oligonucleotide as described above. In one embodiment, the kit includes at least two synthetic oiigonucleotides having different flanking regions, and thus being targeted to different sequences of the RNA substrate. In another embodiment, the kit further includes an RNA ligase.

Yet another aspect of the present invention is a method of enhancing the ribonuclease activity of a synthetic catalytic oligonucleotide. In this method, at least one nucleotide in the 3' or 5' flanking region is modified by substituting a propynyl substituent at position 5 of the nucleotide.

Another aspect of the invention is a method of controlling the expression of a target RNA molecule. In this method, the target RNA is contacted with a synthetic catalytic oligonucleotide of the invention. The 5' flanking region of the synthetic oligonucleotide hybridizes to the first target region of the substrate RNA, and the 3' flanking region of the synthetic oligonucleotide hybridizes to the second target region of the substrate RNA, the 5-propynyl substituent (s) providing increased base stacking and hybridization stability, thereby opyimizing the ability of the synthetic oligonucleotide to more effectively bind and cleave the substrate RNA and then release the products of cleavage. In this way, the conflicting requirements of stable hybridization yet rapid dissociation to achieve high turnover are satisfied, and the expression of the substrate RNA, e.g., its ability to be translated into protein, is controlled. In another aspect of the invention, a method of site-specifically cleaving a single-stranded, RNA-containing substrate is provided. As used herein, the term "site-specifically cleaving" refers to enzymatically cutting the phosphate backbone of the substrate RΝA molecule before or after a particular sequence of ribonucleotides. The method includes contacting the RΝA-containing single-stranded substrate molecule with a synthetic oligonucleotide of the invention such that the 3' flanking region of the synthetic oligonucleotide hybridizes to the first target region of the substrate RΝA, and the 5' flanking region of the synthetic oligonucleotide hybridizes to the second target region of the substrate RΝA molecule thereby enabling the synthetic oligonucleotide to site-specifically cleave the RΝA substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present invention, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

FIG. IA is a schematic representation of 5- propynyl substituted uridine;

FIG. IB is a schematic representation of 5- propynyl substituted deoxy-uridine;

FIG. IC is a schematic representation of 5- propynyl substituted cytidine;

FIG. ID is a schematic representation of 5- propynyl substituted deoxy-cytidine;

FIG. 2 is a diagrammatic representation of a consensus hammerhead ribozyme hybridized with a substrate RNA, wherein the conserved ribonucleotides (C, U, G, A, G, A, G, A, A) and the non-conserved nucleotide (N) are in the catalytic core of the ribozyme, and wherein cleavage occurs on the 3' side of nucleotide (Y) in the substrate RNA; and

FIG. 3 is a graphic representation of the cleavage activity of R46, a catalytic oligonucleotide of the invention, in the presence (—▼—) and absence (—V—) of a facilitator nucleotide (F15) , in comparison with the cleavage activity of R23, a ribozyme control having 5 nucleotides in each of its flanking regions, in the presence (—•—) and absence (—o—) of the same facilitator oligonucleotide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art . The issued U.S. patent, allowed patent applications, and articles cited herein are hereby incorporated by reference.

The present invention provides compositions and methods for controlling gene expression and for enhancing the endonucleolytic or ribonuclease activity of catalytic synthetic oiigonucleotides. Compositions of the invention are synthetic catalytic oiigonucleotides having at least one propynyl substitution in position 5 of a pyrimidine nucleotide. Such substituted pyrimidines include, for example, 5- (1-propynyl) - uridine (FIG. IA) ; 5- (1-propynyl) -2' -deoxyuridine (FIG. IB) ; 5- (1-propynyl) -cytidine (FIG. IC) ; and 5- (1-propynyl) -2' -deoxycytidine (FIG. ID) .

The 5-propynyl-substituted pyrimidine nucleotide is located at least in one region of the catalytic oligonucleotide which is complementary to a sequence on the target molecule to which it hybridizes, and which flanks the specific site on the substrate molecule to be cleaved. Catalytic oiigonucleotides of the invention have two such "flanking regions," one at the 3' terminal portion of the molecule, and one at the 5' terminal portion of the molecule. The flanking regions are from about four to fifteen nucleotides in length, with flanking regions of about five or six being common. More than one pyrimidine nucleotide may be substituted in this way, and all of the pyrimidine nucleotides in the flanking regions of the synthetic catalytic oligonucleotide of the invention may be likewise substituted. Substitutions may be made in a 3' or 5' terminal or penultimate or any other pyrimidine nucleotide in the flanking region(s) .

Catalytic synthetic oiigonucleotides of the invention including such 5-propynyl-substituted pyrimidines include ribozymes, ribozyme analogs, and other molecules containing 3' to 5' covalently linked nucleotides having the ability to endonucleolytically cleave a single-stranded RNA substrate at its phosphate backbone.

Ribozymes may assume one of several physical structures, one of which is called a "hammerhead" (Haseloff and Gerlach (1988) Nature 334:585-591) , and is depicted in FIG. 2. A hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double-stranded stem and loop structure (helix II) , and two regions flanking the catalytic core that are complementary to the target RΝA. The flanking regions enable the ribozyme to bind to the target RΝA specifically by forming double-stranded stems I and III. Cleavage occurs in ώ (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RΝA substrate other than that containing the ribozyme) next to specific ribonucleotide triplet by a transesterification reaction from a 3', 5'-phosphate diester to a 2', 3'-cyclic phosphate diester.

Some of the synthetic oiigonucleotides of the invention are structurally distinct from an unmodified hammerhead ribozyme in that any or all pyrimidine nucleotide (s) in the flanking regions are 5-propynyl substituted. Table 1 lists a number of representative, nonlimiting catalytic oiigonucleotides containing nucleotide substitutions at various locations which are marked with an asterisk.

TABLE 1

SEQ Sequence (5'→3 ' ) ID NO:

A U A C U CUGAUGAGGCCGUUAGGCCGAA A C G C U* 1

A U A C U CUGAUGAGGCCGUUAGGCCGAA A C G C*U 1

A U A C U CUGAUGAGGCCGUUAGGCCGAA A C G C*U* 1

A U A C U CUGAUGAGGCCGUUAGGCCGAA A C*G C*U* 1

A U*A C U*CUGAUGAGGCCGUUAGGCCGAA A C*G C*U* 1 A A U A C U CUGAUGAGGCCGUUAGGCCGAA A C*G C*U* 2

A A U A C U CUGAUGAGGCCGUUAGGCCGAA A C*G C*U*U 2

C*C U A C U CUGAUGAGGCCGUUAGGCCGAA A C G C U 5

C C*U A C U CUGAUGAGGCCGUUAGGCCGAA A C G C U 5

C*C*U A C U CUGAUGAGGCCGUUAGGCCGAA A C G C U 5 C*C*U*A C*U*CUGAUGAGGCCGUUAGGCCGAA A C G C U 5

C*U*A C*U CUGAUGAGGCCGUUAGGCCGAA A C G C U 6

C*C U A C U CUGAUGAGGCCGUUAGGCCGAA A C G C U*U 7

C C*U A C U CUGAUGAGGCCGUUAGGCCGAA A C G C*U U 7 _c*c*U*A C*U CUGAUGAGGCCGUUAGGCCGAA A C*G C*U*U* 7

N* five-propynyl substituted nucleotide flanking regions are bolded

Although facilitator oiigonucleotides may be used with the catalytic oiigonucleotides of the invention, the 5-propynyl oiigonucleotides, alone, have activity.

The synthetic oligonucleotide may also be modified in a number of ways for protection against nuclease digestion, without preventing hybridization of the synthetic catalytic oiigonucleotides of the invention to substrate RNAs. For example, any of the nucleotides in the flanking sequences also may be substituted with other substituents including deoxynucleotides, 2'- 0-alkylated nucleotides, nucleotide methylphosphonates, and nucleotide phosphoramidates. Some preferred substitutions include a 2' -0-alkylated nucleotides such as 2'-0- methyls, 2' -0-propyls, and 2'-0-butyls. The most preferred nucleotide analog is a 2' -O-methyl. The nucleosides of the flanking regions and other nucleotidic portions of the synthetic oligonucleotide may be covalently linked by other than phosphodiester internucleoside linkages between the 5' end of one nucleoside and the 3' end of another nucleoside, in which the 3' phosphate has been replaced with any number of chemical groups. Examples of such known chemical groups include alkylphosphonates, carbamates, phosphorothioates, phosphoramidates, acetamidate, carboxymethyl esters, carbonates, and phosphate esters.

Other modifications include those which are internal or at the end(s) of the flanking region(s) and include additions to the internucleoside phosphate linkages, such as cholesteryl, or diamine compounds with varying numbers of carbon residues between the amino groups, and terminal ribose, deoxyribose, and phosphate modifications. Examples of such modified flanking regions include nucleotide sequences having a modified base and/or sugar such as arabinose instead of ribose, or a 3' , 5'- substituted nucleoside having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than oxygen or phosphate. Other modified nucleotide sequences are capped with a nuclease resistance-conferring bulky substituent or self-hybridized region at their 3' and/or 5' end(s) , or have a substitution in one nonbridging oxygen per nucleotide. Such modifications can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule.

The synthetic catalytic oiigonucleotides can be prepared from commercially obtainable 5- propynyl substituted and unsubstituted nucleotides by art-recognized methods such as phosphoramidate or H-phosphonate chemistry which can be carried out manually or by an automated synthesizer using standard H-phosphonate chemistry as described in U.S. Patent No. 5,149,789, or using standard phosphoramidite chemistry (see, e.g., Beaucage

(Meth. Mol. Biol. (1993) 20:33-61) ; Damha et al . (in Protocols for Oiigonucleotides and Analogs; Synthesis and Properties (Agrawal, ed.) (1993) Humana Press, Totowa, NJ, pp. 81-114); or Uhlmann et al. (Chem. Rev. (1990) 90:534-583) .

The preparation of modified synthetic oiigonucleotides is well known in the art (reviewed in Agrawal et al . ( 1992 ) Trends Biotechnol.

10 : 152 - 158 ; in Goodchild ( 1990 ) Bioconjugate Chem.

2 : 165 - 187 ) ; Zon in Protocols for Oiigonucleotides and Analogs

(Agrawal, ed.) Humana Press, Totawa, NJ (1994) Vol. 20, pp. 165-189) .

The synthetic catalytic oiigonucleotides of the invention can be provided for any method of use in the form of a kit including a container of a synthetic oligonucleotide of the invention, of mixtures of different synthetic oiigonucleotides, and/or of synthetic oligonucleotide (s) and an RNA ligase. The amount of synthetic oligonucleotide in the container may be sufficient for one therapeutic dose or assay. Alternatively, the amounts of the kit constituents may be concentrated such that only small aliquots need be sampled at one time from the container when used, for example, to cleave RNA molecules in vitro . The kits must preserve the synthetic oligonucleotide (s) and RNA ligase in active form. Any RNA ligase capable of covalently joining single stranded RNA molecules containing 5'- phosphate and 3'-hydroxyl termini is useful. One such ligase is bacteriophage T4 RNA ligase.

The present invention also provides therapeutic formulations containing a synthetic oligonucleotide in a form useful for treatment. These therapeutic formulations are administered to individuals in a manner capable of delivering the synthetic oligonucleotide initially into the body and subsequently into any number of target cells. One mode of administration is via a therapeutic formulation which contains at least one synthetic oligonucleotide, as described above, along with a physiologically acceptable carrier. Some therapeutic formulations contain more than one type of synthetic oligonucleotide of the invention.

As used herein, a "physiologically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, and agents which improve oligonucleotide uptake, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The therapeutic formulations of the invention may be administered parenterally, orally, sublingually by inhalation of spray, by intravenous intramuscular, intraocular, intraperitoneal, or other mode of injection, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term

"parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intraperitineal injection or infusion techniques. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacterial and f ngi. The carrier can be a solvent or dispersion medium.

The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents. Prolonged absorption of the injectable therapeutic agents can be brought about by the use of the compositions of agents delaying absorption.

The amount of synthetic oligonucleotide that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dose levels are in the range of 1 μg to 10 mg. However, it will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific composition employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and severity of the particular disease undergoing therapy.

The synthetic oiigonucleotides of the invention, themselves, or in a therapeutic formulation may be administered or utilized for any purpose known to those with skill in the art that a ribozyme may be used. For example, cells infected with a virus may be treated with a synthetic catalytic oligonucleotide having flanking sequences complementary to nucleotide sequences of a particular mRNA corresponding to a viral gene in order to hinder the expression of that gene. Similarly, synthetic oiigonucleotides may be administered to stop the expression of cancer-related genes, or of any gene which is being overexpressed in vitro or in vivo . Synthetic oiigonucleotides are also useful in probing the function of a particular gene in vitro or in vivo, for example, by knocking out its function and observing the result.

Synthetic oiigonucleotides according to the invention are also useful as RNA-specific restriction endonucleases, and as such, in combination with RNA ligases, allow for the preparation of recombinant RNA molecules.

Using the methods described herein, a representative catalytic synthetic oligonucleotide of the invention having a 3'-terminal 2'deoxy- uracil with a 5-propynyl substituent (R46, SEQ ID NO:l) , was synthesized and tested for its nucleolytic activity. The results shown in FIG. 3 demonstrate that the synthetic catalytic oligonucleotide of the invention has enhanced ribonuclease activity relative to control ribozyme R23 (SEQ ID NO:2) having 5 nucleosides in each flanking region, and has comparable activity to the combination of R23 and F15, a facilitator oligonucleotide (SEQ ID NO:3) .

The following examples illustrate the preferred modes of making and practicing the present invention, but are not meant to limit the scope of the invention since alternative methods may be utilized to obtain similar results.

EXAMPLES

1. Synthesis of Oiigonucleotides

Substrate RNA, radiolabelled internally using

[α-³²P]ATP, was prepared as described by Goodchild and Kohli (Arch. Biochem. Biophys. (1991) 284:386-391) using T7 RNA polymerase and chemically synthesized single-stranded templates with a double-stranded promoter (Milligan et al. (1987) Nucleic Acids Res. 15:8783-8798) . Oligodeoxynucleotides were synthesized using standard automated phosphoramidite procedures (Atkinson et al. in Oligonucleotide Synthesis. A Practical Approach (Gait, ed.) IRL Press, Washington, D.C. (1985) pages 35-81) , then purified by polyacrylamide gel electrophoresis.

Concentrations of radiolabelled substrate were determined from the specific activity of the [α-³²P]ATP used for labelling. Concentrations of unlabelled RNA were determined spectroscopically from the absorption at 260 nm. Extinction coefficients at this wavelength were determined from the sum of the coefficients of the component nucleotides allowing for the hypochromicity of the RNA observed when a sample was digested to completion using snake venom phosphodiesterase and bacterial alkaline phosphatase.

Facilitator oiigonucleotides which contain unmodified (phosphodiester-linked) deoxyribonucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems, Foster City, CA) on a 1.0 μmole scale using standard H-phosphonate chemistry as described in U.S. Patent No. 5,149,789, or using standard phosphoramidite chemistry as described by Beaucage (Meth. Mol. Biol. (1993) 20:33-61) or Uhlmann et al. (Chem. Rev. (1990) 90:534-583) .

Synthesis of Synthetic Catalytic Oiigonucleotides

Synthetic catalytic oiigonucleotides were synthesized on a 1 μmol scale using the automated solid-support phosphoramidite method (Usman et al . (1987) J. Amer. Chem. Soc. 109:7845-7854) with 2'-0- silyl nucleoside phosphoramidites and 2'-0-silyl, 5-propynyl pyrimidine phosphoramidites (Glen Research,Sterling, VA) . Products were cleaved from the support and deblocked using concentrated ammonium hydroxide: ethanol (3:1 v/v) at 55°C for 16 hours. The supernatant solution was divided into halves which were processed separately. After evaporation of solvent, each half of the product was dissolved in a solution of tetrabutyl- ammonium fluoride (TBAF) in 1 M tetrahydrofuran (THF) (0.4 ml) and kept in the dark at room temperature for 16-24 hours to remove silyl groups. The solution was cooled in ice and treated with ice cold 50 nM Tris-HCl, pH 7.4 (0.4 ml) . Following addition of loading dye (0.8 ml of 95% formamide in water containing 0.05% by weight of Orange G) , the solution was heated to 95°C, cooled, and applied directly to a polyacrylamide gel for purification by electrophoresis as described for substrate RNA (Goodchild et al . (1991) Arch. Biochem. Biophys. 284:386-391) .

Cleavage Activity Assay

A solution (45 μl) containing a 5-propynyl substituted synthetic catalytic oligonucleotide (R46, SEQ ID NO:l) or a ribozyme control (R22, SEQ ID NO:2) at a final concentration 0.025 μM, substrate RNA (S12, SEQ ID NO:4; final concentration, 0.5 μM) and facilitator oligonucleotide (F15, SEQ ID NO:3) where appropriate (final concentration 1.0 μM) in 50 nM Tris-HCl (pH 7.4) was brought to the reaction temperature for 10 minutes. Reactions were initiated by the addition of 200 mM MgCl₂ (5 μl; final concentration 20 mM) . After the indicated times, aliquots of 5 μl of the reaction were added to 10 μl of loading dye (95% formamide in water containing 0.05% by weight of Orange G) and put on ice. Samples were denatured by heating at 95°C for 2 minutes and analyzed by electrophoresis on 15% polyacrylamide gel containing 8 M urea. Radioactive bands were quantitated using a phosphorimager (Molecular Dynamics, Sunnyvale, CA) . Representative results are shown in FIG. 3.

4. Stability Assay

The following procedure is used to examine the half-life of synthetic oiigonucleotides in human serum. A solution (50 μl) containing 5'- [³²P] end-labelled synthetic oligonucleotide (40,000 - 150,000 cpms) and tRNA (final concentration of 300 μM) in H₂0 is prepared. 5 μl of the solution is removed and added to 20 μl of loading dye (9 M urea, 100 mM EDTA in H₂0) containing 0.05% by weight Orange G) and placed on dry ice. The remaining 45 μl is evaporated to dryness. The resulting pellet is dissolved in 45 μl human serum (Sigma Chemical Co., St. Louis, MO) and incubated at 37°C. At indicated times, aliquots of 5 μl of the reaction are added to 20 μl of loading dye and placed on dry ice. Samples are denatured by heating at 95°C for 2 minutes and analyzed by electrophoresis on 15% polyacrylamide gel containing 8 M urea.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Hybridon, Inc.

(ii) TITLE OF INVENTION: SYNTHETIC OLIGONUCLEOTIDES WITH ENHANCED RIBONUCLEASE ACTIVITY

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Lappin & Kusmer LLP

(B) STREET: 200 State Street

(C) CITY: Boston

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02109

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kemer, Ann-Louise

(B) REGISTRATION NUMBER: 33,523

(C) REFERENCE/DOCKET NUMBER: HYZ-044PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-330-1300

(B) TELEFAX: 617-330-1311

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : AUACUCUGAU GAGGCCGUUA GGCCGAAACG CU 32

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 : AAUACUCUGA UGAGGCCGUU AGGCCGAAAC GCU 33

(2) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 :

CTCGCACCCA TCT 13

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : GCUAGAAGGA GAGAGAUGGG UGCGAGAGCG UCAGUAUUA 39 (2) INFORMATION FOR SEQ ID NO:5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : CCUACUCUGA UGAGGCCGUU AGGCCGAAAC GCU 33

(2) INFORMATION FOR SEQ ID NO: 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 : CUACUCUGAU GAGGCCGUUA GGCCGAAACG CU 32 (2) INFORMATION FOR SEQ ID NO: 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 : CCUACUCUGA UGAGGCCGUU AGGCCGAAAC GCUU 34

Claims

What is claimed is:

1. A synthetic catalytic oligonucleotide having enhanced ribonuclease activity, the synthetic oligonucleotide comprising a 5' flanking region and a 3' flanking region, each flanking region having about four to fifteen nucleosides, at least a portion of the 3' flanking region being complementary to a first target region of a substrate RNA molecule, and at least a portion of the 5' flanking region being complementary to a second target region of the substrate RNA molecule, at least one of the flanking regions comprising a pyrimidine nucleotide with a 5- propynyl substituent.

2. The synthetic oligonucleotide of claim 1 wherein the 3' flanking region comprises a pyrimidine nucleotide with a 5-propynyl substituent.

3. The synthetic oligonucleotide of claim 1 wherein the 5' flanking region comprises a pyrimidine nucleotide with a 5-propynyl substituent.

4. The synthetic oligonucleotide of claim 2 wherein the 5' flanking region comprises a pyrimidine nucleotide with a 5-propynyl substituent .

5. The synthetic oligonucleotide of claim 1 wherein a flanking region comprises two or more pyrimidine nucleotides having a 5-propynyl substituent .

6. The synthetic oligonucleotide of claim 1 wherein the 3' and the 5' flanking regions each comprise two or more pyrimidine nucleotides having a 5-propynyl substituent.

7. The synthetic oligonucleotide of claim 1 wherein the 3' and the 5' flanking regions each have about five to ten nucleosides.

8. The synthetic oligonucleotide of claim 7 wherein the 3' and the 5' flanking regions each have five to six nucleosides.

9. The synthetic oligonucleotide of claim 1 wherein the pyrimidine nucleotide having the 5- propynyl substituent is the 3' terminal nucleotide of the 3' flanking region.

10. The synthetic oligonucleotide of claim 1 wherein the pyrimidine nucleotide having the 5- propynyl substituent is the 5' terminal nucleotide of the 5' flanking region.

11. The synthetic oligonucleotide of claim 1 wherein the pyrimidine nucleotide having the 5- propynyl substituent is the 3' penultimate nucleotide of the 3' flanking region.

12. The synthetic oligonucleotide of claim 1 wherein the pyrimidine nucleotide having the 5- propynyl substituent is the 5' penultimate nucleotide of the 5' flanking region.

13. The synthetic oligonucleotide of claim 1 wherein the pyrimidine nucleotide is a deoxyribonucleotide.

14. The synthetic oligonucleotide of claim 1 wherein the pyrimidine nucleotide is a ribonucleotide.

15. The synthetic oligonucleotide of claim 14 wherein the pyrimidine nucleotide is a 2'-O-methyl ribonucleotide.

16. The synthetic oligonucleotide of claim 1 which is modified.

17. The synthetic oligonucleotide of claim 1 further comprising:

(a) a nucleotidic stem-loop region having a 3' terminus and a 5' terminus and comprising a plurality of 3' to 5' covalently-linked, self-hybridizing nucleotides;

(b) first and second nucleotidic core regions, each comprising a plurality of 3' to

5' covalently-linked nucleotides, and each having a 3' terminus and a 5' terminus, the 3' terminus of the first nucleotidic core region being covalently linked to the 5' terminus of the stem-loop region, and the 5' terminus of the second nucleotidic core region being covalently linked to the 3' terminus of the stem-loop region, the first and second nucleotidic regions forming a catalytic core,

the catalytic core being flanked by the 3' and 5' flanking regions, the 3' and 5' flanking regions each having a 3' terminus and a 5' terminus, the 3' terminus of the first flanking region being covalently linked to the 5' terminus of the first nucleotidic core region, and the 5' terminus of the second flanking region being covalently linked to the 3' terminus of the second nucleotidic core region.

18. A pharmaceutical formulation comprising the synthetic oligonucleotide of claim 1 in a physiologically acceptable carrier.

19. A pharmaceutical formulation comprising at least two oiigonucleotides of claim 1 having different 3' and 5' flanking regions in a physiologically acceptable carrier.

20. A kit comprising the synthetic oligonucleotide of claim 1.

21. A kit comprising at least two synthetic oiigonucleotides of claim 1 having different flanking regions.

22. A kit comprising the synthetic oligonucleotide of claim 1 and an RNA ligase.

23. A method of site-specifically cleaving a target nucleic acid comprising contacting an RNA target molecule with a synthetic catalytic oligonucleotide having enhanced ribonuclease activity, the synthetic oligonucleotide comprising a 5' flanking region and a 3' flanking region, each flanking region having about five to fifteen nucleosides and each flanking region being complementary to specific target nucleic acid nucleotide sequence, at least one of the flanking sequences comprising a pyrimidine residue with a 5-propynyl substituent.

24. A method of controlling the expression of a target RNA molecule, comprising contacting the target RNA with a synthetic catalytic oligonucleotide having enhanced ribonuclease activity, the synthetic oligonucleotide comprising a 5' flanking region and a 3' flanking region, each flanking region having about five to fifteen nucleosides and each flanking region being complementary and hybridizing to specific target nucleic acid nucleotide sequence, at least one of the flanking sequences comprising a pyrimidine residue with a 5-propynyl substituent,

25. A method of enhancing the ribonuclease activity of a synthetic catalytic oligonucleotide, the synthetic oligonucleotide comprising a 5' flanking region and a 3' flanking region, each flanking region having about five to fifteen nucleosides, and each flanking region being complementary to a specific target nucleic acid nucleotide sequence, the method comprising modifying at least one pyrimidine nucleoside in the 3' or the 5' flanking region of the ribozyme by substituting a propynyl substituent at position 5 of the nucleoside.