CA2093664C - Modified ribozymes - Google Patents

Abstract

The present invention refers to an RNA molecule with catalytic activity comprising at least one modified nucleoside, wher-ein the hydroxy group at the 2'-position of the ribose sugar is replaced by a modifier group, selected from halo, sulfhydryl, azido, amino, monosubstituted amino and disubstituted amino groups, a process for the preparation of modified RNA molecules and the use of modified RNA molecules as therapeutic agents and biocatalysts.

Description

i~;~ied Ribczymes SPECIFICATION
Certain naturally occuring ribonucleic acids (RNAs) are subject to self-cleavage. The first reported example is the cleavage of the ribosomal RNA precursor of the protozoan Tetrahymena (for a review see Cech, Ann.Rev.Biochem. 59 (1990), 543-568) which requires guanosine as cofactor. A
number of examples of RNA cleavage have been subsequently discovered in viroid, virusoid and satellite RNAs (for reviews see Sheldon et al. in Nucleic Acids and Molecular Biology (1990) Vol. 4, pg. 227-242, ed. F. Eckstein and D.M.J. Lilley, Springer Verlag Berlin Heidelberg; Symons, TIBS 14 (1989), 445-450). These cleavages involve site-specific breakage of a phosphodiester bond in the presence of a divalent cation such as Mgz+, generating a 5'-hydroxyl and a 2',3',-cyclic phosphodiester terminus. Sequence analysis around the site of self-cleavage of several of such RNAs has led to the identification of a common structural feature essential for cleavage which was named a "hammerhead"
structure (Hutchins et al., Nucleic Acids Res. 14 (1986) 3627-3640). This structure consists of three helices and 13 conserved nucleotides (framed in below scheme) which form a three dimensional structure amenable to cleavage at one ' particular position. The self-catalyzed cleavage is normally an intramolecular process, i.e. a single RNA molecule contains all the functions necessary for cleavage. However, Uhlenbeck (Nature 328 (1987), 596-600) has demonstrated that this hammerhead structure does not nave to be embodied in one strand but can be made up of two strands. These two strands combine to form the hammerhead structure which leads to phosphodiester bond cleavage (indicated by an arrow) in one of the strands (strand S) whereas the other (strand E) remains unaltered and can participate in many cleavage reactions. This strand meets the definitions of an enzyme and is called a ribozyme. Whereas the framed sequences (below scheme) are conserved the others may vary provided that the 2 0 9 3 6 f~ 4 PCT/E1P91/01811r~" .

structure of base paired and the single stranded regions remains intact.
;~ r'.v ' "'. . GPPP
C G
C G
U A
C G S
A U
A C
AGGA U
A
UA C UC CUGGGppp G GCC U
UGCCGG G
. A A
G U
The cleavage reaction after the trinucleotide GUC has been studied in detail (Ruffner et al., Gene 82 (1989), 31-41;
Fedor and Uhlenbeck, Proc.Natl.Acad.Sci. USA 87 (1990), 1668-1672). Ribozymes with new specificities have also been constructed (Haseloff and Gerlach, Nature 334 (1988), 585-591) indicating that cleavage can for example also take place after the sequences GUA, GUU, CUC, AUC and UUC.
Further examples for RNA enzymes are the hairpin RNA (Hampel et al., Nucleic Acids Res. 18 (1990), 299-304), as well as RNA containing proteins such as the telomerase (Greider and Blackburn, Nature 337 (1989), 331-337) and the RNase P (Beer et al., in Nucleic Acids and Molecular Hiology ('1988), Vol.

3, pp. 231-250, ed. F. Eckstein and D.M.J. Lilley, Springer Verlag, Berlin/Heidelberg).
Ribozymes are potentially of interest for use as therapeutic agents (for review see Rossi and Sarver, TIBTECH 8 (1990), 179-183). A possible strategy would be to destroy an ANA
necessary for the expression of both foreign genes such as WO 92/07065 ~ Q ~ 3 ~ ~ ~ PGT/EP91/01811 viral genes and particular endogenous genes. This requires the construction of a RNA molecule which is able to form a hammerhead ar a hairpin structure with the target RNA and to cleave this at a predetermined position. A first application to the inhibition of the HIV-1 virus by this strategy has been reported (Sarver et al., Science 247 (1990), 1222-1224).
Other examples of the action of targeted hammerhead ribozymes in vivo are Cammeron and Jennings (Proc.Natl.Acad.Sci. USA 86 (1986), 9139-9143) and in vitro Cotten et al. (Mol.Cell.Biol.
9 (1989), 4479-4487).
Further, other useful catalytic properties of ribozymes are known, e.g. dephosphorylase and nucleotidyl transferase activities (see Patent Application W088/04300). Therein RNA
enzymes are disclosed which are capable of dephosphorylating oligonucleotide substrates with high sequence specifity, which distinguishes them from known protein enzymes. RNA
molecules also can act as RNA polymerases, differing from protein enzymes in that they use an internal rather~than an external template. Thus, various heteropolymers can be constructed by variant RNA enzyme forms. This enables the formation for example of messenger RNA molecules for particular proteins or peptides. Furthermore, Herschlag and Cech (Nature 344, (1990), 405-409) describe an RNA enzyme with DNase acitivity.
To be useful as a therapeutic agent the RNA enzyme has to be introduced into target cells. There are a priori two methods for delivery of the ribozyme into the target cells:
(a) exogenous delivery of a preformed synthetic'RNA;
(b) endogenous transcription of a ribozyme-coding gene located on a plasmid.
A great disadvantage of method (a) resides in the very low stability of RNA molecules under physiological conditions due to their fast degradation by a variety of ribonuclPase WO 92/07065 ~ ~ ~ J ~ a ~ pGT/EP91/O1~1L.._ enzymes present in the living cell. The disadvantages of method (b) result from the great difficulties of specifically and stably inserting a ribozyme-coding gene into the cells of higher organisms. Furthermore, the problem of degradation also occurs with in vivo synthesized RNA molecules.
Therefore the problem underlying the present invention was to provide RNA molecules comprising both catalytic activities and enhanced stability against chemical and enzymatical degradation, which can be employed as therapeutical agents or as biocatalysts in biochemical or biotechnological processes.
It was however known from a recent paper by Perreault et al.
(Nature 344 (1990), 565-567) that certain modifications of the RNA enzyme, e.g. the incorporation of 2'-deoxyribo-nucleotides at a few positions of a ribozyme lead to a great impairment of the catalytic activity.
It was now surprisingly found that certain chemical modifications at the 2'-position of the ribose sugar Which enhance the stability of an RNA molecule do not considerably affect and/or abolish the catalytic properties of ribozymes.
Therefore it is an object of the present invention to provide an RNA molecule with catalytic activity comprising at least one modified nucleoside, wherein the hydroxy group at the 2'-position of the ribose sugar is replaced by a modifier group, selected from halo, sulfhydryl, azido, amino, mono-substituted amino and disubstituted amino groups.
The catalytic activity of an RNA molecule according to the present invention comprises advantageously at least one of the group consisting of nucleotidyl transferase, dephosphorylase, deoxyribonuclease and sequence specific endoribonuclease activities. Preferably the catalytic WO 92/07065 ~ ~ ~ ~ ~ ~ ~ p(T/EP91/01811 S
activity comprises a sequence specific endoribonuclease activity. More preferably the RNA is a hammerhead ribozyme as described above. Especially preferred is that the ribozyme can combine with another RNA strand to form a hammerhead structure consisting of two strands, wherein the modified RNA
strand is the E strand as described above.
Although a hammerhead ribozyme is especially preferred, other RNA enzymes are encompassed also by the present invention, e.g. the Tetrahymena ribozyme (Cech, Ann.Rev.Biochem. 59 (1990), 543-568) in naturally occuring form or a shortened form thereof (Zang et al., Biochemistry 27 (1988), 8924 -8931), and especially the Hairpin RNA (Hampel et al., Nucleic Acids Res. 18 (1990) 299-304) or RNA containing proteins such as the RNase P (Baer et al., in Nucleic Acids & Molecular Biology (1988), Vol. 3, pp 231-250, ed. F. Eckstein and D.M.J. Lilley, Springer Verlag Heidelberg), the telomerase (Greider and Blackburn, Nature 337 (1989), 331-337).
The incorporation of a modifier group at the 2'-position of the ribose sugar appears also to be particularly useful for RNA with new functions either derived at by a procedure that depends on alternate cycles of selection (Tuerk and Gold, Science 249 (1990), 505-510; Ellington and Szostak, Nature 346 (1990), 818-822) or any other method .
The modifier group replacing the hydroxy group at the 2'-position of the ribose sugar is selected from halo, sulfhydryl, azido, amino, monosubstituted amino, and disubstituted amino groups. The halo group can be a fluoro, chloro, bromo or iodo group, wherein the fluoro group is preferred. The substituents of the substituted amino group are preferably C~-C3 alkyl and or hydroxyalkyl groups. Most preferably the modifier group is a halo or an unsubstituted amino group.

WO 92/07065 ~ ~ 9 PGT/EP91/0181,1 C
The incorporation of a modifier group at the 2°-position of the ribose sugar significantly increases the RNA stability against enzymatic cleavage. It was confirmed that 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-aminouridine incorporated at specific positions of a ribozyme prevented cleavage at these positions by RNase A (see Fig. 3 + 4). This enzyme cleaves at the 3'-position of pyrimidine nucleosides and requires the presence of the 2'-hydroxyl group (Uchida and Egami (1971), in The Enzymes Vol. IV, 3rd ed. (Ed. P.D. Boyerj, Academic Press, pp. 205-250). Furthermore, results obtained with polynucleotides show that the presence of the 2'-amino function also slows down degradation by unspecific nucleases such as snake venom phosphodiesterase (Flobbs et al., Biochemistry 12 (1973), 5138-5145). The presence of a 2'-halogroup also inhibits nucleases such as DNase I (Hobbs et al., Biochemistry 11 (1972), 4336-4344). Results with polynucleotides also show that the presence of a halogen at the 2'-position of a nucleotide protects against the action of human serum nucleases (Black et al., Virology 4 8 (1972) 537-545). Thus, protection by incorporation of a modified ribose sugar according to the present invention will be rather general and not. be restricted to RNases which depend on the presence of the 2'-hydroxyl group. .
In a ribonucleic acid the ribose sugar is linked to a nucleotide base via a N-glycosidic bond. The nucleotide base, which is attached to the modified ribose sugar in an RNA
molecule of the present invention is selected from the group consisting of bases naturally occuring in RNA and substituted bases. Preferably the modified ribose is attached to adenine, guanine, cytosine and/or uracil, which are the natural bases in RNA. The modified ribose, however, can also be attached to substituted bases, preferably selected from the group consisting of xanthine, hypoxanthine, 2,6-diamino purine, 2-hydroxy-6-mercaptopurine and purine bases substituted at the 6-position with sulfur or pyrimidine bases substituted at the 5-position with halo or C~-C5 alkyl groups, especially bromo or methyl groups. Most preferably the nucleotide base attached to the modified ribose sugar is uracil.
The modified nucleosides which are incorporated into a RNA
molecule are either previously described compounds or compounds which can be prepared in analogy to known compounds. The mostly preferred fluoro and amino analogs of ribonucleosides have been described previously, 2'-deoxy-2'-fluorocytidine (Doerr & Fox, J.Org.Chem. 32 (1967), 1462;
Mengel & Guschlbauer, Ang.Chem. 90 (1978), 557-558); 2'-deoxy-2'-fluoroadenosine (Ikehara & Miki, Chem.Pharm.Bull. 26 (1978), 2449-2453), 2'-deoxy-2'-fluorouridine (Codington et al., J.Org.Chem. 29 (1964), 558-564), 2'-deoxy-2'-aminouridine (Verheyden et al., J.Osg.Chem. 36 (1971), 2S0) and 2'-deoxy-2-aminocytidine (Verheyden et al. (1971) supra).
For the synthesis of some of these compounds more recent synthetic procedures can be employed. The 2'-deoxy-2'-fluorocytidine can be prepared from 2'-deoxy-2'-fluorouridine by the method of Sung (J.Org.Chem. 47 (1982), 3623-3628). The same method can be used for the transformation of 2'-deoxy-2°-azidouridine to 2'-deoxy-2'-azidocytidine (Verheyden et al. (1971), supra). The latter can be reduced to 2'-deoxy-2'-aminocytidine by the method of Mungall et al. (J.Org.Chem. 40 (1975), 1659).
The synthesis of the 2'-deoxy-2'-fluoronucleoside 5'-triphosphates can be carried out either according to Zudwig (Acta Biochim. et Biophys. Acad.Sci.Eung. 16 (1981), 131-133) or Ludwig and Eckstein (J.Org.Chem. 54 (1989), 631-635). The 2'-deoxy-2'-aminouridine and -cytidine 5'-triphosphates can be prepared as described for the diphosphates by Hobbs et al.
(Biochemistry 12 (1973), 5138-5145) with the modification that pyrophosphate is employed instead of phosphate. The 2'-deoxy-2'-fluoronucleoside 3'-phosphoramidites for automated oligonucleotide synthesis can be prepared by the method of WO 92!07065 PGT/EP91/01811 2093G~~ v a Sinha et al. (Nucleic Acids Res. 32 (1984), 4539-4557). F'or the synthesis of the corresponding 2'-amino derivatives, the amino group can be protected by~trifluoroacetylation according to Imazawa and Ecks'tein (J.Org.Chem. 44 (1979), 2039-2041).
An RNA according to the present invention comprises at least one modified nucleoside; wherein the hydroxy group at the 2'-position of ribose is replaced by a modifies group. A
preferred embodiment of the present invention is an RNA
molecule wherein all nucleosides of one kind (i.e. adenosine or guanosine or cytidine or uridine) contain modified sugars, while the remaining three nucleosides contain unmodified sugars. More preferably the modified nucleoside is pyrimidine nucleoside, i.e. cytidine or uridine or a substituted derivative thereof. Most preferably the modified sugar is 2'-fluoro ribose or 2'-amino ribose. Examples for this 1 embodiment are the hammerhead ribozymes E2 and E3, which were derived from a hammerhead ribozyme E1 described by Fedor and Uhlenbeck (Proc.Natl.Acad.Sci. USA 87 (1990), 1668-1672). In E2 all uridine residues are replaced by 2'-deoxy-2'-fluoro-uridine and in E3 all uridine residues are replaced by 2'-deoxy-2'-aminouridine residues. The ribozymes E2 and E3 show a ribonuclease activity which is comparable to that of the unmodified RNA molecule E1.
In a further preferred embodiment of the present invention all nucleosides of two different kinds contain modified sugars, while the remaining two nucleosides contain unmodified sugars. More preferably all pyrimidine nucleosides, i.e. cytidine and uridine (including substituted pyrimidine bases) contain modified sugars, most preferably 2'-fluoro or 2'-amino ribose derivatives.
Still a further embodiment of the present invention is an RNA
molecule comprising a modification pattern (i.e. which WO 92/07065 ~ ~ ~ ~ ~ ~ /~ PGT/EP91/01811 nucleosides are modified and which are unmodified) which is designated as a so-called "selective modification pattern".
An RNA comprising selective modification pattern is a molecule wherein nucleosides at specifically selected locations can be modified while nucleosides at other specifically selected locations can be unmodified. For instance, nucleotides which are known to be hypersensitive sites for ribonucleases (e. g. due to the secondary structure of the RNA molecule) should be modified to achieve an extended life time of the RNA molecule. An example for a ribonuclease-hypersensitive site is provided at position 21 of ribozyme E1. As shown in Fig. 3 the RNA molecule is cleaved at this position by RNase A with very high intensity.
Still a further embodiment of the present invention is a RNA
molecule additionally comprising at least one modified internucleotidic,phosphodiester linkage. Examples for suitable modified phosphodiester linkages are methyl phosphonate groups or phosphorothioate groups, the latter being especially preferred. Preferably at least the 5'-terminal phosphodiester linkage and/or the 3'-terminal phosphodiester linkage of the RNA molecule is modified. More preferably the 5'-terminal phosphodiester linkage and the last three 3'-terminal phosphodiester linkages are modified.
It was found, that the presence of modified internucleotidic linkages alone was not sufficient to provide increased stability against degradation. However, the combined presence of 2'-modified ribose sugars together with modified internucleotidic linkages showed an additive stability enhancing effect. A more than fiftyfold increase in stability confered by both modifications outweighs the decreased efficiency in cleavage compared to a unmodified ribozyme.

WO 92/07065 ~ ~ ~ J a ~ ~ P~'/EP91/0181~~~.
/o The synthesis of RNA molecules having modified inter-nucleotidic linkages~is preferably accomplished by means of chemical synthesis as described below.
A further object of the present invention is a process for the synthetis of an RNA molecule with catalytic activity, comprising:
incorporating into an RNA chain at least one modified nucleoside, wherein the hydroxy group at the 2'-position of the ribose sugar is replaced by a modifier group, selected from halo, sulfhydryl, azido, amino, monosubstituted amino and disubstituted amino groups.
Preferably the modifier group is a halo (i.e. a fluoro, chloro, bromo or iodo group) or an amino group, more preferably a fluoro or an unsubstituted amino group. It' should be noted, that the process of the present invention also comprises the synthesis of an RNA molecule wherein nucleotides with at least two different modifier groups (e. g.
fluoro and amino groups) are incorporated.
There are preferably two approaches for the incorporation of these modified nucleotides into RNA. One is by automated chemical synthesis of RNA molecules which can be carried out on solid support or in solution, preferably with the respective phosphoramidites or H-phosphonates as nucleotide precursors, the other involves enzymatic incorporation by transcription of appropriate nucleic acid, preferably DNA
templates With a nucleic acid polymerase using the 2'-modified nucleoside 5'-triphosphates. Hy means of automated chemical synthesis RNA molecules comprising modified internucleotidic linkages may be prepared by incorporating the corresponding chemically modified nucleotide precursors such as the methyl phosphonate derivatives into the RNA
chain. For the incorporation of phosphorothioate linkages the standard phosphoramidite derivatives are used as nucleotide WO 92/07065 ~ ~ ~ ~ ~ ~ ~ pCT/FP91/01811 precursors. After the coupling of the precursor to the RNA
chain has taken place the subsequent oxidation step, however, is not performed with iodine, as in the case of non-modified linkages, but with sulfur or a sulfurating agent, whereby the phosphorothioate group is obtained.
The chemical synthesis of modified RNA molecules is carried out in analogy to that of unmodified RNA or DNA molecules, which is known in the art. More specifically the RNA
synthesis is carried out by chemical synthesis on solid support involving the stepwise addition of the respective nucleotide precursors. After having synthesized an RNA
product of the desired length, the RNA is removed from the solid support by conventional means and purified, preferably by gel electrophoresis. Alternatively the chemical RNA
synthesis can also be carried out by any other known technique without using a solid support. E.g. the RNA can be synthesized in a soluble form and subsequently purified by means known in the art.
When the 2'-amino modifier group is incorporated into the RNA
chain it has to be protected before the phosphitylation reaction (i.e. the preparation of the nucleotide precursor) ' and for subsequent use in the coupling reactions. For this purpose the trifluoroacetyl group is preferably used as a protecting group, because it is stable during the cycles of synthesis on the nucleic acid synthesizer and is removable under the conventional treatment with ammonia.
Alternatively the synthesis of the RNA chain can~be carried out by transcription from a nucleic acid template by an appropriate nucleic acid polymerase. Preferably the template is a DNA template and the nucleic acid polymerase is a DNA
dependent RNA polymerase. More preferably the DNA dependent RNA polymerase is selected from the group consisting of T7, T3 and SP6 polymerases, which are highly processive WO 92/07065 ~ ~ ~ p~yFp91/01811 ~ ' Iz bacteriophage RNA polymerises. Among these polymerises the T7 RNA polymerise is most preferred. The DNA template for the synthesis of a modified RNA molecule according to the present invention is preferably constructed by inserting a synthetic ' DNA fragment coding for the desired RNA sequence into an appropriate site of a plasmid, wherein said plasmid comprises a promoter for the respective RNA polymerise and said site is ' located at such a position of the plasmid, so that the synthetic DNA fragment can be transcribed from said promoter.
The transcription reaction is advantageously carried out as a run off transcription. Alternatively, synthetic DNA fragments may serve as transcription templates without a plasmid portion. Those fragments, however, should contain a transcription start signal, which allows an effective RNA
synthesis.
The polymerisation of 2'-deoxy-2'-halo nucleotides, e.g. 2'-deoxy-2°-fluorouridine, -cytidine, -adenosine, -guanosine and the respective chloro compounds, is preferably carriEd out by T7 polymerise in the presence of Mn2' ions as cofactor.
Alternatively, the polymerisation of 2'-aminonucleotides, e.g., 2'-deoxy-2'-aminouridine 2'-deoxy-2'-aminocytidine, 2'-deoxy-2'-aminoadenosine, and 2°-deoxy-2'-aminoguanosine, is ' preferably carried out in the presence of Mg2° ions as cofactor.
From the experimental data of the following examples it is evident that the presence,of 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-aminouridine in a hammerhead ribozyme do not abolish catalytic activity. This is qualitatively shown in Fig. 3 for the presence of the 2'-fluorouridine in the substrate part and quantitatively in Table 1 for various other enzyme/substrate,pairs. It is true that all the modifications resulted in an increase in the ~ -value which was most pronounced for the amino substitution. However, this perturbation of the active structure lies well within the WO 92/07065 ~ ~ ~ ~ ~ ~ ~ PCT/EP91/01811 range of Km variation observed for hammerhead systems with different base composition (Fedor & Uhlenbeck, supra). In addition, very surprisingly the incorporation of a single aminouridine immediately 5' of the site of cleavage in the substrate increased the kcat markedly (table 1), so that it is conceivable to produce ribozymes of enhanced activity by the selective introduction of 2'-modified nucleosides at specific sites. These results definitely show that there is no requirement for the presence of 2-hydroxyl groups throughout the enzyme part of the hammerhead structure for catalytic activity but that the modifications according to the present invention are tolerated at least in certain positions. In contrast, the incorporation of only 15 % 2'-deoxynucleotides into a hammerhead ribozyme is reported to decrease the catalytic efficiency by two orders of magnitude, while not affecting the R", (Perreauit et al. (1990), supra).
Since the rate of cleavage is determined by the angle of attack of the 2'-hydroxyl on the phosphorus at the site of cleavage, it is greatly influenced by the overall s$ructure of the hammerhead system. Thus, the observed influence of 2'-modifications on the rate supports the notion that the 2'-fluoro analogs adopt a structure more similar to that of ribonucleotides than that of deoxyribonucleotides. This apparently also holds fox the amino analogs. The other 2'-modified nucleosides according to the present invention exhibit similar catalytic activity.
A still further object of the present invention is the use of RNA molecules with catalytic activity comprising at least one modified nucleotide, as therapeutic agents, especially for the specific cleavage of viral or other foreign genetic material or transcripts from viral or other foreign genetic material, or as biocatalyst in biochemical or biotechnological processes. For these purposes the RNA
molecules of the present invention seem to be more suitable than their unmodified analogs, because of their increased stability against chemical and/or enzymatical cleavage.

WO 92/07065 2 Q ~ 3 ~ 6 ~ PGT/EP91/01811...._ ~4 The present invention shall be further illustrated by the following examples in.combination with Figs. 1 - 7. These examples however are~not intended to narrow the scope of the present invention.
Fig. 1 shows autoradiographs of T7 RNA polymerise run off transcripts of the plasmid pUCRS after PAGE.
Fig. 2 shows an autoradiograph of T7 RNA polymerise run off transcripts of the plasmid pUCRE containing 2'-aminouridine after PAGE.
Fig. 3 shows an autoradiograph of partial Ribonuclease A
cleavage of 5'-labeled.run off transcripts E1 and E2 separated by PAGE.
Fig. 4 shows an autoradiograph of the total degradation of S1 and S2 by RNase A.
Fig. 5 shows an autoradiograph of the cleavage of~2'-fluorouridine and 3zP-AMP containing substrate S3 by ribozyme E1.
Fig. 6 shows an Eadie-Hofstee plot of the ribozyme reaction of E2 with S1.
Fig. 7 shows an Lineweaver-Burk plot of the ribozyme reaction of E3 with S1.
Fig. 8 shows the organisation of the HIV-1 sequence cloned into pOTH33. .
Fig. 9 shows the nucleotide sequence of the ribozyme RE115.

_ - 15 -EXAMPIrES
Example 1 Preparation of oligoribonucleotides Automated synthesis of oligoribonucleotides: Automated oligoribonucleotide synthesis was carried out with an Applied Biosystems 3808 DNA Synthesizer on a 1 umol scale using the monomeric ribonucleotide phosphoramidites supplied by Milligen/Biosearch. Control pore glass columns with the ribonucleoside coupled to it were either from Milligen/Bio-search or Peninsula. The oligomers were worked up according to the specifications of the supplier of the ribonucleotide phosphoramidites (Milligen/Biosarch). After removal of the protecting groups the oligoribonucleotides were concentrated by spin dialysis on Amicon filter membranes centricon*'10 and ethanol precipitated. The dried pellets were taken up in 50 u1 water and subjected to PAGE. Bands were visualized by UV shadowing, cut out and the RNA was isolated by eluting at 37°C overnight in buffer (0.25 M ammonium acetate, 10 mM
TRIS/HC1 pH 8.0, 1 mM EDTA) (Fedor & Uhlenbeck, PNAS USA 87 (1990), 1668-1672). Concentrations were determined using the extinction coefficient per nucleotide of 6600 M'~cm'~ given in the literature (Fedor & Uhlenbeck 1990). Aqueous solutions of the oligoribonucleotides were stored at -20°C.
Construction of plasmids containing templates for run off transcription:
The following oligodeoxynucleotides were synthesized for the plasmid construction by the phosphoramidite method with an Applied Biosystems 380B DNA synthesizer:
RS2-T,5'-d(GATATCCTGACTCCCTATAGTGAGTCGTATTA)-3; RS2-C,5'-d(TAATACGACTCACTATAGGGAGTCAGGATATCTGCA)-3'; REl-T,5'-d(GGAGTTTCGGCCTAACGGCCTCATCAGAGGACCCTATAGTGAGTCGTATTA)-3' and RE2-C,5'-d(TAATACGACTCACTATAGGGTCCTCTGATGAGGCCGTTAGGCCGAA.ACTCCTGCA)-3'.
* trade-mark Preparation of ribozyme pUCRS and pUCREI6 clones:
The commercially available plasmid pUCl9 was cleaved in a one step reaction using the restriciton enzymes Iso-SacI and PstI. The DNA was then purified by 2 $ agarose gel electrophoresis followed by electroelution using a Centricon*
30 and the centroelution apparatus supplied by Amicon. The oligonucleotide primer pairs, RE1-T and RE2-C (ribozyme enzyme), or RS2-T and RS2-C (ribozyme substrate) were phosphorylated as previously described (Taylor et al., Nucleic Acids Res. 13 (1985), 8749-8764). These oligonucleotide pairs were used for cloning of the T7 promotor along with either the DNA sequence for the ribozyme yielding pUCREI6 or the ribozyme substrate yielding pUCRS
according to the pracedure of King & Blakesley (Focus 8 (1986), 1-3). After transformation of competent cells (Olsen & Eckstein, PNAS USA 87 (1990), 1451-1456) white colonies were screened for the presence of a second AvaII site in the case of the pUCREI6 or a unique EcoRV site for pUCRS. The sequence of the purified double-stranded DNA from each clone was determined by the procedure of Olsen and Eckstein (Nucleic Acids Res. 17 (1989), 9613-9620).
T7 RNA polymerase run off transcripts:
T7 RNA polymerase run off transcripts were synthesized on a 150 ~1 to 500 ~tl scale by adapting the procedure given by Milligan and Uhlenbeck (Meth. in Enzymology 180A (1989), 51-62). Transcription reactions were run in 40 mM TRIS pH 8.0, 1mM spermidine, 5 mM DTT, 0.01 ~ Triton*x-100, 20 mM MgCl2, 2.5 mM nucleotides, 200 nM DNA template, 0.2 U/ul human placental RNase inhibitor, and 100 U/~.rl T7 RNA polymerase.
When 2'-deoxy-2'-fluoronucleoside triphosphates were employed, the MgCl2 was replaced by 20 mM MnCl2. Reactions were run at 37°C for 3 hours. Transcripts were purified by PAGE as described above. Aqueous solutions of the oligoribonucleotides were stored at -20°C.
* trade-marks WO 92/07065 ~ ~ PCT/EP91/01811 r~
Figure 1 shows autoradiographs of T7 RNA polymerase run off transcriptions of pUCRS after PAGE. As The transcription was performed on a 150 pL scale in the presence of 20 mM MgClz and 2.5 mM each of the four nucleoside triphosphates at 37°C
for 3 h. The reaction mixture was dephosphorylated with alkaline phosphatase and 5'-32P-labeled by reaction with polynucleotide kinase and [x-32P]-ATP. The labeled transcription mixture was subjected to PAGE. B: The transcription was performed on a 150 ;rL scale at 37°C for 3 h in the presence of 20 mM MnClZ , 0.5 mM ATP, 25 ~rCi [a-3 2 P]-ATP, 2.5 mM CTP and GTp, and 2.5 mM 2'-fluorouridine triphosphate. The transcription mixture was directly subjected to PAGE. The asterisks mark 3ZP-labeled phosphates.
'N' denotes any nucleotide added by T7 RNA polymerase beyond the full length of the template DNA (c.f. Milligan and Uhlenbeck, Meth. in Enzymology 180A (1989), 51-62).
Figure 2 shows an autoradiograph of T7 RNA polymerase run off transcripts of pUCRE 16 containing 2'-aminouridine after PAGE. xane 1: 2'-aminouridine containing 34-mer marker E3, synthesized chemically. Lane 2: The transcription was performed on a 150 ;t1 scale at 37°C for 3 h in the presence ' of 20 mM MgCl2, 60 uCi [a-32PJATp, 1 mM CTP and GTP, and 1 mM
2'-aminouridine triphosphate. The transcription mixture was dizectly applied PAGE.
Preparation of oligoribonucleotides: The following oligoribonucleotides were prepared a.) by run off transcription (sequences given without the 5'-triphosphate):
5'-GGGUCCUCUGAUGAGGCCGUUAGGCCGAAACUCC-3';
~,5'-GGG(2'-FU)CC(2'-FU)C(2'-FU)GA(2'-FU)GAGGCCG(2'-FU)(2'-FU)AGGCCGAAAC(2'-FU)CC-3' and ~5'-GGG(2'-NHpU)CC(2'-NH2U)C(2'-NH2U)GA(2'-NH2U)GAGGCCG(2'-NH2 U ) ( 2 ' -NHZ U ) AGGCCGAAAC ( 2 ' -NH2 U ) CC-3 ' ;

WO 92/07065 2 O ~ ~ PCT/EP91101811_ !8 S1.5'-GGGAGUCAGGAU-3'; S3.5'-GGGAG(2'-FU)CAGGA(2'-FU)-3' and S4.5'GGGAGU(2'-FC)AGGAU-3' b.) by chemical synthesis: The oligoribonucleotides E1, E2, E3, S1 and ~, 5'-GGGAG(2'-NHZU)CAGGAU-3'.
5'-3ZP-labeling of oligoribonucleotides:
Oligoribonucleotides obtained from run off transcriptions were dephosphorylated by treatment with RNAse free bovine alkaline phosphatase, purified by Quiagen tip-20 columns according to the protocol given by the manufaturer (Diagen Inc.) and treated with T4 polynucleotide kinase and ~-32p-ATP. Labeled oligoribonucleotides were purified by PAGE.
Example 2:
Digestion of oligoribonucleotides with RNase A
Partial digestion of oligoribonucleotides with RNase A: The oligosibonucleotides E1 and E2 were subjected to RNase A
digestion after 5'-32P labeling according to the procedure of Donis-Kelley et al. (Nucleic Acids Res. 4 0977), 2527-2538) with the following changes. Approximately 25 ~rmoles of 5'- ' 32P-labeled RNA was added to 50 ~1 buffer containing 7 M
urea, 50 mM EDTA, 0.04 % bromophenol blue, 0.04 % xylene cyanol FF and 0.25 mg/ml tRNA on ice. The RNA was then equally divided into 5 labeled tubes, heated to 50°C for 5 min and then immediately placed on ice. Ribonuclease A, 2 girl (2 X 10'4 units), was added to the first tube and mixed using the pipette. The enzyme was then successively 5. fold diluted into three additional tubes using a new pipette tip after each transfer from one tube to the next. The fifth tube was a control sample to which no enzyme was added. All tubes were then incubated at 50°C for 5 min, placed on ice and analysed by PAGE. w WO 92/07065 2 ~ ~ ~ ~ ~ ~~ PGT/EP91/01811 Total degradation of oligoribonucleotides by RNAse A: The oligoribonucleotides S1 and S2 were digested with RNase A
after 5'-3ZP labeling according to the following protocol:
The oligomer (8.5 ;rM in a final volume of 20 u1) was reacted with 1.25 x 10'3 Units of RNAse A in buffer containing 50 mM
TRIS/HCl pH 7.5 and 10 mM MgCl2 for 10 min at 37°C. Products were analyzed by PAGE.
Figure 3 shows an autoradiograph of partial Ribonuclease A
cleavage of 5'-labeled run off transcripts E1 and E2 separated by PAGE. Conditions as described before. The numbered lanes correspond to 1) no enzyme added, 2) 2x10-4 units RNase A, 3) 3x10'5 units RNase A, 4) 8x10'6 units RNase A, 5) 16x10~~ units RNase A. Hase numbering was facilitated by counting the bands of a Mnz* mediated cleavage of the unmodified transcript (10 Irmoles RNA heated to 90°C for 3 min in 10 mM MnCl2). The circled numbers indicate the bands expected from RNase-A susceptible cleavage positions. Arrows indicate the bands that arise from cleavage 3' to a=idine and which are absent in the lanes where 2'-fluorouridine containing ribozyme was cleaved.
Figure 4 shows an autoradiograph of the total degradation of~
S1 and S2 by RNase A after PAGE. Details of the reaction are as described above. bane 1: total digestion of 12-mer S2;
lane 2: total digestion of 12-mer Sl; lane 3: cleavage ladder of the 34-mer E1 by reaction with 20 mM MnCl2 at 90°C for 3 min as a length standard. The product of cleavage of S2 is 1 nucleotide longer than that of S1 indicating the presence of 2'-aminouridine at position 6.

Example 3 Cleavage of oligoribonucleotide substrates by ribozymes Determination of cleavage kinetics: The cleavage reactions were performed by a procedure adapted from Fedor and Uhlenbeck (1990), supra). Stock solutions of the ribozyme enzyme (typically 20 uL final volume, 100 nM final concentration, 50 mM TRIS/HCl, pH 7.5) and substrate oligonucleotide (typically 40 u1, 2 ACM final concentration) were heated separately at 90°C for 1 min and cooled to room temperature for 15 min prior to the addition of divalent metal ion (MnClZ or MgCl2, 10 mM final concentration). These stocks were incubated separately at 25°C for 15 min prior to initiation of the cleavage reactions. The reactions were started by adding enzyme to substrate (50 mM TRIS/HC1, pH
7.5, 20 N1 final volume, MgCl2, 10 mM final concentration), with typical concentrations of IO nM enzyme and between 50 and 5000 nM substrate. At set times 10 ~Cl aliquots were transferred into IO u1 urea stop mix and subjected to PAGE.
Autoradiographs were analyzed on an LKB ULTROSCA~*XZ laser densitometer.
In the investigated hammerhead ribozyme system a I2-mer substrate oligonucleotide (designated as S) is cleaved by a 34-mer enzyme oligonucleotide (designated as E) at the 3'-position of cytidine-7 as indicated by the arrow in the structure in the Introduction. This cleavage generates a heptamer with a 2'-3'-cyclic phosphate terminus (product 1) and a pentamer with a 5'-hydroxyl terminus (product 2) (Ruffner et al, Gene 82 (1989), 31-41). We observed these types of cleavage products not only with the oligoribo-nucleotides EI and S1, but also with the 2'-fluorouridine-containing substrate S3 (Fig.5). As expected, the substrate oligonucleotide S4, containing a 2'-fluorocytidine at the position of cleavage was not cleaved under identical conditions. These two reactions contained 2'-fluorouridine in the substrate oligonucleotide.
* trade-mark WO 92/07065 ~ Q ~ j ~ ~ '3 PCT/EP91/01811 2~
However, potentially more interesting for future applications is the question whether the presence of this modification in the enzyme part of the ribozyme will interfere with its catalytic activity. Thus, the reaction of the 2'-fluorouridine-containing ribozyme E2 with the unmodified substrate S1 was investigated. Indeed, the gel analysis indicated that the substrate was cleaved with similar efficiency as the pair E1 and S1. The catalytic constants of the 2'-fluorouridine-containing ribozyme~E2 were determined (Fig. 6) and compared to those of the unmodified ribozyme El.
This comparison reveals that the second older sate constant for the former (k~lt/Rm = 0.0026 nM'~) is one order of magnitude smaller than that of the latter (k~at/K", _ 0.023 nM'~) (Fedor & Uhlenbeck (1990), supra) (Table 1). This decrease in catalytic efficiency is primarily due to a decrease in the rate of cleavage, whereas the Rm values for both ribozymes is nearly identical. This reduced rate of cleavage, however, lies well within the range of cleavage efficiencies observed for various hammerhead systems with different base compositions (Fedor & Uhlenbeck (1990), supra). Hammerhead ribozyme reactions can be carried out with MgCla as well as with MnCl2 as metal ion cofactor, Where the half life of cleavage is decreased in the presence of the latter cofactor by about 10 fold (Uhlenbeck, Nature 328 (1987), 596-609). Such a decrease in the half life of the substrate under cleavage conditions upon switching from Mg2+
to Mn2+ was also observed for the reaction of 2'-fluorouridine-containing enzyme E2 with substrate S1. Thus the~metal ion requirement for the cleavage reaction is not altered by the incorporation of 2'-fluoronucleotide analogs.
The effect of the presence of 2'-aminouridine in the ribozyme was also investigated. When the 2'-aminouridine containing ribozyme E3 is reacted with nonmodified substrate S1, the catalytic efficiency drops an order of magnitude to k~at/Rm 0.0015 nM'~. This decrease in efficiency is clearly due to a WO 92/07065 ~ ~ ~ ~ ~ ~ ~ PGT/EP91 /0181 ~.- , z2 higher Km, while the k~ac remains almost unaltered. Thus, the overall efficiency of the 2'-aminouridine ribozyme is comparable to the one of the 2'-fluorouridine containing ribozyme. In the complementary reaction of the nanmodified ribozyme E1 with the selectively,2'.-aminouridine modified substrate S2 the catalytic efficiency is increased compared to the above reaction to k~et/Km = 0.0063 nM-~. This effect is entirely due to an increase in kcat. This trend is even more pronounced in the reaction of the 2'-aminouridine containing ribozyme E3 with S2, where the catalytic efficiency is increased to kGat/Km = 0.011 nM'~, again mainly due to an increased k~et~ The kinetic parameters for all of the above reactions are summarized in Table 1.
Table 1:
Kinetic constants of 2'-modified nucleotide-containing ribozymes.e Enzyme Substrate k~ a s Km kc s t /Km (~n~' ) (nM) (nM' ~ min' ~ ) E1(nonmod.) S1 (nonmod.)3.0 140 0.023 E2(2-FU) S1 (nonmod.)0.8 300 0.0026 E3(2-NHZU) S1 (nonmod.)2.3 1500 0.0f15 E3(2'-NH2U) S2 (2'-NHZU)19.0 1800 0.011 E1(nonmod.) S2 (2'-NH2U)10.0 1600 0.0063 Kinetic constants were determined from Eadie-Hofstee plots of cleavage reactions sun with 10 nM ribozyme and with substrate concentrations ranging from 50 nM to 1200 nM.
Thus,.the herein compiled kinetic data shows that while the cleavage efficiency of 2'-fluoro- and 2'-aminouridine modified ribozyme is somewhat reduced, it is still within the WO 92/07065 ~ 0 9 3 ~ ~ ~~ PCT/E~1/01811 range of variations observed for hammerhead systems of different base composition. It also becomes evident that it is possible to increase the catalytic efficiency by selectively introducing 2'-modifications at specific positions. While the latter effect was demonstrated for the substrate oligoribonucleotide, it is anticipated that a similar influence on. catalysis can be found for selective modifications in the enzyme.
Figure 5 shows an autoradiograph of the cleavage of 2'-fluorouridine and 3zP-AMP--containing substrate S3 by ribozyme E1. The cleavage reaction was performed in the presence of 10 mM MgCl2 in 50 mM TRIS/HC1, pH 7.5 on a 40 u1 scale at 25°C.
The concentration of E1 and S3 was 2.5 pM and 7.5 ;rM, respectively. All other details are as described above (c.f.
Determination of Cleavage Kinetics). At the indicated times u1 aliquots were transferred into 10 N1 water and 10 u1 urea stop mix prior to PAGE. Y~ane 1: reaction after-0.5 min;
lane 2: reaction after 15 min; lane 3: reaction after 30 min.
The asterisks mark 32P-labeled phosphates.
Figure 6 shows an Eadie-Hofstee plot of the ribozyme reaction of E2 with S1. The cleavage reaction were performed on a ;r1 scale in the presence of 10 mM MgCl2 with a 10 nM
concentration of E2 and concentrations of S1 of 50 nM, 100 nM, 200 nM, 400 nM, 500 nM, and 700 nM. After 7 min 10 N1 aliquots were transferred into 10 N1 water and 10 Nl urea stop miac prior to PAGE. It was established previously that these time points fall within the linear range of initial velocities. The autoradiographs were evaluated by integration of their optical density on a laser densitometer.
Figure 7 shows an Lineweaver-Burk plot of the ribozyme reaction of E3 with S1. The cleavage reactions were performed on a 20 u1 scale in the presence of 10 mM MgClz with a 10 nM

concentration of E3 and concentrations of S1 of 50 nM, 100 nM, 200 nM, 400 nM, 500 nM and 700 nM. All other details are as in Fig. 6.
Example 4 Cleavage of HIV-1 LTR RNA using ribozymes Plasmid Construction: A plasmid, pOTH33, was constructed by cloning the HIV-1 sequence from position -525 to 386 (according to the sequence numbering by Ratner et al., Nature 313 {1985), 277-284) into the commercially available plasmid pSPTl9 (Pharmacia). The HIV sequence is under transcriptional control of a T7 promotor (T7). A diagrammatic view of the HIV.
insertion in pOTH33 is given in Fig. 8. The HIV-1 LTR region consists of the U3 region, the R region and the U5 region. It is flanked on its 5'-end by the polypurine tract and on its 3'-end by the primer binding site (PBS), the leader sequence and a part of the gag gene. The arrows at position -525 and 386 indicate the restriction sites used for the construction of pOTH33. The arrow at position 115 shows the site for ribozyme mediated cleavage.
RNA of HIV-1 from position -525 to 386 comprising the long terminal repeat sequence from nucleotide -453 to 182 was obtained by run-off transcription of EcoRI cleaved plasmid pOTH33 (100 ng/ul DNA template, 10 mM DTT, 500 uM of each rNTP, 50 mM Tris-C1 pH 7.5, 2 mM spermidine, 6 mM MgCl2, 2 uCi/ul (a-3zP]-ATP, 50 U/ul RNase inhibitor and 15 U/~1 T7 RNA polymerase, 2 h at 4°C) and subsequent incubation of the reaction mix with DNaseI (1 U/ul, 10 min at 37°C) (RNase free) and phenol-chloroform extraction. The obtained RNA was designated as LTR RNA.
Position 115 of the HIV-1 LTR RNA containing the potential cleavage site GUC was chosen as a target for ribozyme catalyzed cleavage. Hammerhead ribozymes targeted against this site were chemically synthesized. The nucleotide sequence of the unmodified hammerhead enzyme RE115 is given in Fig. 9.
Cleavage Kinetics with LTR RNA: k~at/Km values were determined under single turnover conditions. Ribozymes were preincubated at 75°C for 1 min in the presence of 50 mM Tris-C1 pH 7.5 followed by 5 min of incubation at 37°C. MgCl2 was added to a final concentration of 10 mM and the solutions were again incubated for 5 min at 37°C. LTR RNA was directly used as an aqueous solution. The reaction mixture (10 u1) contained between 20 nM and 1 uM ribozyme, 50 mM Tris-C1 pH
7.5 and 10 mM MgCIZ. The reaction was started by addition of LTR RNA to a final concentration of 10 nM. After 1 hour at 37°C the reaction was stopped by addition of 10 u1 stop mix and analysed by 4 $ PAGE (40 cm long, 8 M urea). After 1 h electrophoresis at 50 W followed by autoradiography the fraction of noncleaved LTR RNA was determined by laser scanning densitometry. k~et/Km values were obtained by plotting the remaining fraction of LTR RNA (Frac S) against the ribozyme concentration ([RE]) according to the following equation:
ln(FracS) k~et k ° - [RE]
t Km , where k is the observed reaction rate and t is the reaction time of 1 h.
In order to investigate the influence of chemical modifications on the catalytic efficiency of a ribozyme several analogs of RE115 containing 2'-fluoro or 2'-deoxy substitutions and/or terminal phosphorothioate linkages were synthesized. Whereas 2'-fluorocytidine substitutions had no WO 92/07065 ~ ~ ~ j ~ ~ ~ PCT/EP91/0181~-, z6 effect on the catalytic efficiency [Table 2, RE115(FC)], 2'-fluorouridine substitutions caused a fivefold decrease of kcat~K", (Table 2, RE115(FU)). One 5'-terminal phosphoro-thioate group in combination with three 3'-terminal phosphorothioate groups diminished the catalytic efficiency only negligibly [Table 2, RE115(S)]. The same was true for the combination of terminal phosphorothioate linkages together with 2'-fluorouridine substitutions, where no further decrease in kcet~le", was observed [Table 2, RE115(FU),S)]. Substituting all pyrimidine ribonucleotidse by their 2'-fluoro analogs and introducing the four phosphoro-thioate linkages decreased the catalytic efficiency only sevenfold compared to the unmodified ribozyme [Table 2, RE115(FC,FU,S)). In contrast substitutions of all pyrimidine ribonucleotides by their 2'-deoxynucleoside analogs combined with phosphorothioates resulted in a decrease of kcat~Rm by a factor of 50 [Table 2, RE115(dC,dU,S)). Thus, RE115(dC, dU,S) is some 7 times less efficient than RE115(FC,FU,S).

Influence of chemical modifications on the Cleavage of L,TR
RNA by RE115 RiboZyme kc a t ,gym ~ kc a t ~Rm r M- ~ s- ~ relative RE115(S) 360 0,72 RE115(FC)~ 490 0,98 RE115(FU)~ 89 0,18 RE115(FU,S)~ 59 0,12 RE115(FC,FU,S)~ 69 0,14 , RE115(dC,dU,S)Z 10 0,020 Examples of the present invention Z Comparative example Example 5 Stability of oligoribonucleotides The ribozymes of Example 4 were examined for their stability against nuclease digestion.
Test conditions:
Molt 4 clone 8 cells (kindly supplied by E. Jurkiewicz, Deutsches Primatenzentrum, Gottingen) grown in medium RMPI
1640 to a cell density of about 106 cells/ml were centrifuged at 1000 g for 5 min in a Heraeus Minifuge.*5'-32P-labeled ribozymes were pre-heated for 1 min at 90°C, chilled on ice, added to the cell supernatant to a final concentration of 300 nM and incubated at 37°C. Aliquots were taken at the indicated time points and analysed by 20 ~ PAGE containing 8 M urea followed by autoradiography.
Results:
More than 80 $ of RE115 was degraded after 2 min incubation in the cell supernatant as indicated by denaturing PAGE. For RE115{S) similar results were obtained. However, no degradation of RE115{FC,FU,S) within 1 hour was observed. A
comparison with the rate of degradation of the unmodified ribozyme indicates that the combination of 2'-modified pyrimidine nucleosides and terminal phosphorothioate linkages results in an estimated increase of more than fiftyfold of ribozyme stability against digestion by nucleases present in T cell supernatant. 2'-modified ribozymes without phosphorothiate group show a stability which is about two times lower than the stability of RE115 (FC,FU,S).
* trade-mark

Claims (43)

C L A I M S

1. An RNA molecule with catalytic activity comprising at least one modified nucleoside, wherein the hydroxy group at the 2'-position of the ribose sugar is replaced by a modifier group, selected from halo, sulfhydryl, azido, amino, monosubstituted amino and disubstituted amino groups.

2. RNA according to claim 1, wherein the modifier group is a halo or an amino group.

3. RNA according to claim 1 or 2, wherein the halo group is a fluoro group.

4. RNA according to claim 1, wherein the catalytic activity comprises at least one of the group consisting of nucleotidyl transferase, dephosphorylase, deoxyribo-nuclease, and sequence specific endoribonuclease activities.

5. RNA according to claim 4, wherein the catalytic activity comprises a sequence specific endoribonuclease activity.

6. RNA according to claim 5, wherein it is a hammerhead ribozyme or a hairpin RNA.

7. RNA according to any of the preceding claims, wherein the nucleotide base attached to the modified ribose sugar is selected from the group consisting of bases naturally occuring in RNA and substituted bases.

8. RNA according to claim 7, wherein the substituted nucleotide base is selected from the group consisting of xanthine, hypoxanthine, 2,6-diamino purine, 2-hydroxy-6-mercaptopurine and purine bases substituted at the 6-position with sulfur or pyrimidine bases substituted at the 5-position with halo or C1-C5 alkyl groups.

9. RNA according to claim 7, wherein the nucleotide base attached to the modified ribose sugar is a base naturally occuring in RNA.

10. RNA according to claim 9, wherein the nucleotide base attached to the modified ribose sugar is a pyrimidine base.

11. RNA according to any of the preceding claims, wherein all nucleotide bases of one specific kind are attached to a modified ribose sugar.

12. RNA according to claim 11, wherein all uracil nucleotide bases are attached to a modified ribose sugar.

13. RNA according to claim 11, wherein all cytosine nucleotide bases are attached to a modified ribose sugar.

14. RNA according to any one of the claims 1 - 10, wherein all nucleotide bases of two specific kinds are attached to a modified ribose sugar.

15. RNA according to claim 14, wherein all cytosine and uracil nucleotide bases are attached to a modified sugar.

16. RNA according to any of the claims 11-15, wherein the modified ribose sugar is comprising a 2'-fluoro or a 2'-amino group.

17. RNA comprising the nucleotide sequence E2:
5'-GGG(2'-FU)CC(2'-FU)C(2'-FU)GA(2'-FU)GAGGCCG
(2'-FU)(2'-FU)AGGCCGAAAC(2'-FU)CC-3' wherein 2'-FU represents 2'-deoxy-2'-fluorouridine monophosphate.

18. RNA comprising the nucleotide sequence E3:
5'-GGG(2'-NH2U)CC(2'-NH2U)C(2'-NH2U)GA(2'-NH2U) GAGGCCG(2'-NH2U)(2'-NH2U)AGGCCGAAAC(2'-NH2U)CC-3' wherein 2'-NH2U represents 2'-deoxy-2'-aminouridine monophosphate.

19. RNA according to any one of the claims 1 to 10, comprising a selective modification pattern based on the structural characteristics of the molecule, wherein nucleotides at hypersensitive sites for ribonucleases are modified.

20. RNA according to any one of the claims 1 to 19, further comprising at least one modified internucleotidic phosphodiester linkage.

21. RNA according to claim 20, wherein the modified phosphodiester linkage is a phosphorothioate group.

22. RNA according to claim 20 or 21, wherein at least the 5'-terminal phosphodiester linkage is modified.

23. RNA according to, any one of the claims 20 to 22, wherein at least the 3'-terminal phosphodiester linkage is modified.

24. RNA according to any one of the claims 20 to 23, wherein the 5'-terminal phosphodiester linkage and the last three 3'-terminal phosphodiester linkages are modified.

25. A method for the synthesis of an RNA molecule with catalytic activity, comprising: incorporating into an RNA chain at least one modified nucleotide, wherein the hydroxy group at the 2'-position of the ribose sugar is replaced by a modifier group, selected the group consisting of halo, sulfhydryl, azido, amino, monosubstituted amino and disubstituted amino groups.

26. The method according to claim 25, wherein the modifier group is a halo or an amino group.

27. The method according to claim 26, wherein the halo group is a fluoro group.

28. The method according to claim 26, wherein the synthesis of the RNA
chain is carried out by chemical synthesis from nucleotide precursors on solid support, removing the RNA chain produced from said solid support and purifying the removed RNA chain produced.

29. The method according to claim 26, wherein the synthesis of the RNA
chain is carried out by chemical synthesis from nucleotide precursors in solution and purifying the RNA chain produced.

30. The method according to claim 28 or 29, wherein the nucleotide precursors are phosphoramidites or H-phosphonates.

31. The method according to any one of the claims 28 to 30, wherein the amino modifier group is incorporated in to an RNA chain in form of a trifluoroacetyl amido group, and the trifluoroacetyl amido group is subsequently removed by treatment with ammonia.

32. The method according to any one of the claims 25 to 31, further comprising: incorporating into an RNA chain at least one modified internucleotidic phosphodiester linkage.

33. The method according to claim 32, wherein the modified phosphodiester linkage is a phosphorothioate group.

34. The method according to any one of the claims 25 to 27, wherein the synthesis of the RNA chain is carried out by transcription from a nucleic acid template by a nucleic acid polymerase.

35. The method according to claim 34, wherein the nucleic acid template is a DNA template and the nucleic acid polymerase is a DNA dependent RNA
polymerase.

36. The method according to claim 35, wherein the DNA dependent RNA
polymerase is selected from the group, consisting of T7, T3 and SP6 polymerase.

37. The method according to claim 36, wherein the DNA dependent RNA
polymerase is T7 polymerase.

38. The method according to any one of the claims 34 to 37, wherein the modifier group is halo group and the synthesis of the RNA chain is carried out in presence of Mn2+ ions.

39. The method according to any one of the claims 34 to 37, wherein the modifier group is a amino, monosubstituted amino, or disubstituted amino group and the synthesis of the RNA chain is carried out in presence of Mg2+
ions.

40. Use of an RNA according to any one of the claims 1 to 24 as a therapeutic agent or a biocatalyst.

41. A therapeutic composition comprising an RNA according to any one of claims 1 to 24, and at least one of a filler, adjuvant, carrier and diluent.

42. The method according to claim 27, wherein the synthesis of the RNA
chain is carried out by chemical synthesis from nucleotide precursors on solid support, removing the RNA chain produced from said solid support and purifying the removed RNA chain produced.

43. The method according to claim 27, wherein the synthesis of the RNA
chain is carried out by chemical synthesis from nucleotide precursors in solution and purifying the RNA chain produced.