This invention provides catalytic molecules capable of cleaving target nucleotide sequences. More specifically, the invention provides an endonuclease having nucleotide sequences which are of sufficient length to allow hybridisation to a target nucleotide sequence desired to be cleaved. The endonuclease contains a catalytic region comprising ribonucleotides and/or deoxyribonucleotides, or derivatives thereof which act to cleave a phosphodiester bond of the substrate nucleotide sequence. The catalytic region comprises nucleotides or derivatives thereof which are linked by linking groups which may comprise ribonucleotides, deoxyribonucleotides or combinations thereof. The endonucleases of the invention are useful in the cleavage of target RNAs associated with disease in humans and animals and in the inactivation of RNA transcripts in eukaryotic and prokaryotic cells, as well as the cleavage of RNA transcripts in-vitro.

This invention generally relates to endonucleases which are capable of 
cleaving nucleic acids; vectors encoding endonucleases; host cells and 
organisms either modified by and/or containing or encoding nucleic acid 
endonucleases; and to methods for the cleavage and/or inactivation of 
nucleic acid molecules in-vivo or in-vitro. 
FIELD OF THE INVENTION 
There have previously been described catalytic endoribonucleases comprised 
of RNA which are capable of effecting the cleavage of target RNA. Such 
endoribonucleases generally fall into two categories. The first, are based 
on the mitochondrial intervening sequence (IVS) RNA of the organism 
tetrahymena, such as described by Zaug et al. (Nature, Vol. 324, 429-433, 
1986). The second class of endoribonucleases are the result of pioneering 
work by Haseloff and Gerlach (Nature, Vol. 334, 585-591, 1988) on the 
self-cleaving regions of plant viral RNA. 
This invention is directed to hitherto unknown endonucleases.

SUMMARY OF THE INVENTION 
In accordance with a first aspect of this invention, there is provided an 
endonuclease of the formula (I) 
EQU X--M--Y (I) 
wherein X and Y represent nucleotide sequences comprised of 
deoxyribonucleotides, ribonucleotides, or combinations thereof or 
derivatives thereof; said nucleotide sequences being of sufficient length 
to allow hybridization to a target nucleic acid sequence desired to be 
cleaved by said endonuclease; 
and wherein M represents a catalytic region of the formula (II): 
EQU 5' C.sup.1 U.sup.2 G.sup.3 A.sup.4 N.sup.5 G.sup.6 A.sup.7 -P-G.sup.8 
A.sup.9 A.sup.10 A.sup.11 N.sup.12 3' (II) 
where, A, G, C and U respectively represent bases adenine, guanine, 
cytosine and uracil which may be in the form of deoxyribonucleotides, 
ribonucleotides, or combinations thereof or derivatives thereof; and 
N.sup.12 is selected from any of the bases adenine, guanine, cytosine, 
uracil or thymine, or derivatives thereof; 
and wherein; 
P is one or more nucleotides, which nucleotides may be 
deoxyribonucleotides, ribonucleotides, or a combination of one or more 
deoxyribonucleotides and one or more ribonucleotides, or derivatives 
thereof, wherein if said nucleotides solely comprise ribonucleotides and 
the nucleotide sequences X and Y are solely comprised of ribonucleotides, 
then said ribonucleotides of the group P are not base paired to one 
another; or a bond or any atom or any group of interconnected atoms 
linking nucleotides A.sup.7 and G.sup.8, which does not destroy the 
cleavage capability of the catalytic region and which is not solely 
comprised of nucleotides. 
For the sake of convenience, such endonucleases may be referred to as 
minizymes. 
In the catalytic region M, nucleotide additions, deletions or replacements 
may be made with the proviso that endonuclease activity is not destroyed. 
For example, any one of nucleotides C.sup.1 to A.sup.11 may be substituted 
with one or more ribo- and/or deoxyribonucleotides containing bases such 
as adenine, guanine, cytosine, methyl cytosine, uracil, thymine, xanthine, 
hypoxanthine, inosine, or other methylated bases. Nucleotide bases and 
deoxynucleotide bases are well known in the art and are described, for 
example in Principles of Nucleic Acid Structure (Ed, Wolfram Sangar, 
Springer-Verlag, N.Y. 1984) which is incorporated herein in its entirety 
by reference. Nucleotides C.sup.1 to N.sup.12 may be substituted with any 
ribonucleotide or deoxyribonucleotide known per se, with the proviso that 
endonuclease activity, particularly endoribonuclease activity, is not 
lost. Endoribonuclease activity may be readily and routinely assessed as 
will be described hereinafter. 
It is preferred that the catalytic region be comprised of ribonucleotides. 
Notwithstanding this, one or more of the nucleotides C.sup.1 to A.sup.11 
of the catalytic region may be in the form of deoxyribonucleotides as long 
as endoribonuclease activity is not lost. 
Ribonucleotide and deoxyribonucleotide derivatives or modifications are 
well known in the art, and are described, for example, in Principles of 
Nucleic Acid Structure (Supra, particularly pages 159-200), and in the CRC 
Handbook of Biochemistry (Second edition, Ed, H. Sober, 1970) which is 
incorporated herein by reference. 
Nucleotides comprise a base, sugar and a monophosphate group. Accordingly, 
nucleotide derivatives or modifications may be made at the level of the 
base, sugar or monophosphate groupings. 
A large number of modified bases are found in nature, and a wide range of 
modified bases have been synthetically produced (see Principles of Nucleic 
Acid Structure and CRC Handbook of Biochemistry, Supra). For example, 
amino groups and ring nitrogens may be alkylated, such as alkylation of 
ring nitrogen atoms or carbon atoms such as N.sub.1 and N.sub.7 of guanine 
and C.sub.5 of cytosine; substitution of keto by thioketo groups; 
saturation of carbon.dbd.carbon double bonds, and introduction of a 
C-glycosyl link in pseudouridine. Examples of thicketo derivatives are 
6-mercaptopurine and 6-mercaptoguanine. 
Bases may be substituted with various groups, such as halogen, hydroxy, 
amine, alkyl, azido, nitro, phenyl and the like. 
The sugar moiety of the nucleotide may be modified according to well known 
methods in the art (see Principles of Nucleic Acid Structure and CRC 
Handbook of Biochemistry, Supra). This invention embraces various 
modifications to the sugar moiety of nucleotides as long as such 
modifications do not abolish cleavage activity of the endonuclease. 
Examples of modified sugars include replacement of secondary hydroxyl 
groups with halogen, amino or azido groups; 2'-methylation; conformational 
variants such as the O2'-hydroxyl being cis-oriented to the glycosyl 
C.sub.1' -N link to provide arabinonucleosides, and conformational isomers 
at carbon C.sub.1' to give .alpha.-nucleosides, and the like. 
The phosphate moiety of nucleosides is also subject to derivatisation or 
modifications, which are well known in the art. For example, replacement 
of oxygen with nitrogen, sulphur or carbon derivatives to respectively 
give phosphoramidates, phosphorothioates and phosphonates. Substitutions 
of oxygen with nitrogen, sulphur or carbon derivatives may be made in 
bridging or non bridging positions. It has been well established from work 
involving antisense oligonucleotides that phosphodiester and 
phosphorothioate derivatives may efficiently enter cells (particularly 
when of short length), possibly due to association with a cellular 
receptor. Methylphosphonates are probably readily taken up by cells by 
virtue of their electrical neutrality. 
Deoxyribonucleotide or ribonucleotide derivatives as referred to in this 
specification embody one or more of the modifications referred to above 
which do not destroy the cleavage capability of the endonuclease. 
Bases and/or nucleotides 1 to 11 of the catalytic region may be substituted 
with other chemical species, such as an amino-acid side chain or linkers 
which may or may not incorporate other chemical entities, e.g. acidic or 
basic groups. For example, G.sup.3 may be substituted with tyrosine, and 
C.sup.1 or A.sup.11 similarly substituted with histidine. In some 
instances it may prove possible to delete a functionally important 
chemical species (e.g., nucleotide or amino-acid side chain) and provide 
this as part of the substrate or as a co-factor which transactivates the 
endonuclease. Such derivatives which possess endonuclease activity are 
within the scope of the present invention. 
Endonuclease activity is readily, simply and routinely tested by incubating 
the endonuclease with its substrate and thereafter assessing whether 
cleavage of a the substrate takes place. For example, cleavage of a target 
mRNA takes place after the trinucleotide sequence X'UY' where X' and Y' 
represent any ribonucleotide, and which may be the same or different and U 
represents a ribonucleotide having the base uridine. Preferred cleavage 
sites include GUC, GUU, GUA and UUC. By way of example, suitable reaction 
conditions may comprise a temperature from about 4.degree. C. to about 
60.degree. C. (preferably about 20.degree. to 55.degree. C.), pH from 
about 7.0 to about 9.0 and salt (such as Mg.sup.2+) from about 1 to about 
100 mM (preferably 1 to 20 mM). Endonucleases containing a small number of 
nucleotides in each of the groups X and Y of formula (I) (such as four 
nucleotides) in each of groups X and Y would generally be incubated at 
lower temperatures, such as about 20.degree. C. to about 25.degree. C. to 
aid duplexing of complementary nucleotide sequences in the endonuclease 
sequences X and Y and the substrate. The endonuclease would generally be 
in an equimolar ratio to the substrate or in excess thereof. However, as 
the endonuclease may act as an enzyme, cleaving substrate without 
consumption, the ratio of endonuclease to substrate is not of importance. 
A target RNA containing a suitable cleavage site as mentioned above, such 
as GUC site may be incubated with an endonuclease which, for example, may 
contain one or more modifications within the catalytic region. The 
nucleotide sequences X and Y of the formula (I) are selected so as to be 
complementary (that is, capable of forming base pairs) to nucleotide 
sequences flanking the cleavage site in the target RNA. On incubation of 
the endonuclease and its substrate an enzyme/substrate complex is formed, 
as a result of base pairing between complementary nucleotides in the 
endonuclease and the substrate. Nucleotide sequences X and Y of the 
formula (I) and nucleotide sequences flanking the cleavage site in the 
substrate form a double stranded duplex as a result of base pairing, which 
base pairing is well known in the art (Sambrook, J. et al., Molecular 
Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, 1989, 
which is incorporated herein by reference). The formation of a double 
stranded duplex between complementary nucleotides may be referred to as 
hybridization (Sambrook, et al., Supra). Hybridization or duplex formation 
between the endonuclease and its substrate can be readily assessed, for 
example, by labelling one or both components, such as with a radiolabel, 
and then subjecting the reaction mixture to polyacrylamide gel 
electrophoresis under non-denaturing conditions (Sambrook et al., Supra). 
If the target is cleaved on incubation with the endonuclease it is active 
and is within the scope of this invention. Accordingly, an endonuclease 
containing a nucleotide derivative may be simply tested for endonuclease 
activity in a routine manner. 
As will be readily appreciated by workers in the field to which this 
invention relates, the cleavage of a target RNA may be readily assessed by 
various methods well known in the art (for example, see Sambrook et al., 
Supra). Cleavage may, for example, be assessed by running the reaction 
products (where the substrate is radioactively labelled) on acrylamide, 
agarose, or other gel systems, and then subjecting the gel to 
autoradiography or other analytical technique to detect cleavage fragments 
(Sambrook et al., Supra). 
Where the group P represents a bond it may represent a chemical bond or 
atom or group of interconnected atoms between nucleotides A.sup.7 and 
G.sup.8. The bond between nucleotides is between the base or sugar moiety, 
i.e., sugar to sugar, base to sugar, or base to base. Inter base 
crosslinking is described, for example, by Petric et al. (1991), Nucleic 
Acid. Res., 19:585, which is incorporated herein by reference. The bond 
may extend from any position on the base or sugar ring or from any 
functional group on the base or sugar ring, again with the proviso that 
the endonuclease is capable of substrate cleavage. 
The group P of the formula (II) may represent one or more nucleotides, 
which nucleotides may be deoxyribonucleotides, ribonucleotides, or a 
combination of one or more deoxyribonucleotides or one or more 
ribonucleotides, or derivatives thereof, wherein said derivatives are as 
described herein. Where P comprises a nucleotide sequence, the nucleotide 
sequence may comprise a ribonucleotide sequence or a combination of one or 
more ribonucleotides and one or more deoxyribonucleotides, in which none 
of these bases are paired, or at least one of the bases is base paired 
(i.e., this including where all of the bases are base paired, and where 
not all of the bases are paired). Alternatively, the group P may comprise 
a deoxyribonucleotide sequence in which none of the bases are paired or at 
least one of the bases are paired (again this including where all of the 
bases are base paired, and where not all of the bases are base paired). 
Where the nucleotide sequences X and Y are comprised solely of 
ribonucleotides, and the group P is comprised solely of ribonucleotides, 
the ribonucleotides of the group P are not base paired to one another. 
Optionally, where nucleotide sequences X and Y are comprised solely of 
deoxyribonucleotides, and the group P is comprised solely of 
deoxyribonucleotides, the deoxyribonucleotides of the group P may not be 
base paired. 
Applicants have found that base pairing in the group P is not required for 
cleavage of a target RNA. Accordingly, when nucleotide sequences X and Y 
are comprised solely of ribonucleotides and the group P is comprised 
solely of ribonucleotides, the ribonucleotides of the group P may be base 
paired for purposes other than to effect cleavage of a target RNA. Such 
purposes would include to allow the binding of cellular factors, such as 
RNA binding proteins or other cellular factors. Similarly, where the 
nucleotide sequences X and Y are comprised solely of deoxyribonucleotides, 
and the group P is comprised solely of deoxyribonucleotides, the 
deoxyribonucleotides of the group P may be base paired for purposes other 
than involvement in endonuclease cleavage, such as interaction with DNA 
binding proteins and other cellular factors, which may, for example, 
effect cellular distribution of the endonuclease. 
Where deoxyribonucleotides, ribonucleotides, combinations thereof, and 
derivatives thereof, are said to be not base paired, a person of ordinary 
skill in the art will understand this to mean that the nucleotides are not 
base paired to one another according to known nucleotide base pairing, 
namely Watson-Crick base pairs and Hoogsteen base pairs, and the like 
(Principles of Nucleic Acid Structure, Supra). 
The absence of base pairing is an advantageous feature of this invention as 
endonucleases comprising a minimal number of nucleotides may be produced 
according to standard methods hereinafter described. The applicants have 
surprisingly discovered that base pairs between nucleotides in the group P 
are not required to permit the endonuclease of this invention to cleave 
its target substrate. Accordingly, the group P may comprise any number of 
non base paired nucleotides, for example, two nucleotides (such as TT), 
four nucleotides (such as AAAA, UUUU, TTTT, etc.) or five nucleotides 
(such as TTTTT). The nucleotide sequence of the group P under these 
circumstances is not of importance and the number of nucleotides is also 
not of importance. Generally speaking, the group P may comprise from 1 to 
20 non base paired nucleotides and more preferably may contain from 2 to 6 
non base paired nucleotides. The main consideration to take into account 
is that the resultant endonuclease is capable of substrate cleavage. This 
can be readily measured without undue experimentation in standard cleavage 
assays on an appropriate target nucleotide sequences as hereinbefore 
described. 
The group P in formula (II) may comprise ribonucleotides, 
deoxyribonucleotides, or at least one deoxyribonucleotide and at least one 
ribonucleotide, or derivatives thereof wherein all nucleotides are base 
paired, or not all of the nucleotides are base paired, as previously 
described. Complementary nucleotides in the nucleotide sequence P linking 
nucleotides A.sup.7 and G.sup.8 of the formula (II), may be base paired by 
Watson-Crick base pairs, Hoogsteen base pairs, or other base pairing known 
in the art. Where the nucleotide sequence of the group P is partly base 
paired (that is, not all nucleotides are base paired), there may be 
provided regions of base pairing and one or more single-stranded regions, 
for example, a base paired stem and a loop of non base paired nucleotides. 
For example, a stem and loop arrangement is described by Haseloff and 
Gerlach (Supra), and has the following sequence: 
##STR1## 
A sequence forming such a stem/loop structure may, in accordance with this 
invention, be comprised of deoxyribonucleotides, ribonucleotides, or a 
combination of deoxyribonucleotides and ribonucleotides. 
Notwithstanding the above, it is preferred that the nucleotides of the 
group P are not base paired. The absence of a base paired structure may 
reduce steric interference of the endonuclease with its substrate or other 
nucleic acid sequences (thus increasing endonuclease activity), and may 
reduce the likelihood of non-favourable base pairing interactions. 
The group P of the formula (II) may comprise a nucleotide sequence as 
herein described wherein one or more ribonucleotides and/or 
deoxyribonucleotides are replaced with a linker which connects adjacent 
nucleotides. Any chemical linker, that is any group of interconnected 
atoms, may be used to link nucleotides A.sup.7 and G.sup.8, or any one of 
nucleotides C.sup.1 to A.sup.7 with any one of nucleotides G.sup.8 to 
A.sup.10 wherein the endonuclease is capable of substrate cleavage. 
Examples of such linkers are described by Petric et al., Supra). Substrate 
cleavage may be readily assessed by simply incubating an endonuclease with 
its substrate as described hereinbefore. 
One or more ribonucleotides and/or deoxyribonucleotides of the group P may 
be replaced, for example, with a linker selected from optionally 
substitited polyphosphodiester (such as poly(1-phospho-3-propanol)), 
optionally substituted alkyl, optionally substituted polyamide, optionally 
substituted glycol, and the like. Optional substituents are well known in 
the art, and include alkoxy (such as methoxy, ethoxy and propoxy), 
straight or branch chain lower alkyl (such as C.sub.1 -C.sub.5 alkyl), 
amine, aminoalkyl such as amino C.sub.1 -C.sub.5 alkyl), halogen (such as 
F, Cl and Br) and the like. The nature of optional substituents is not of 
importance, as long as the resultant endonuclease is capable of substrate 
cleavage. 
Additionally, suitable linkers may comprise polycyclic molecules, such as 
those containing phenyl or cyclohexyl rings. Such compounds would 
generally comprise suitable functional groups to allow coupling through 
reactive groups on nucleotides. 
The nucleotides of the groups X and Y may be of any length and sequence 
sufficient to enable hybridization formation with complementary 
nucleotides in the target RNA, as described herein. The nucleotides may be 
in the form of deoxyribo-nucleotides, ribonucleotides, deoxyribonucleotide 
ribonucleotide hybrids, or derivatives thereof as hereinbefore described. 
These flanking sequences may be chosen to optimize stability of the 
endonuclease from degradation. For example, deoxyribonucleotides are 
resistant to the action of ribonucleases. Modified bases, sugars or 
phosphate linkages of nucleotides, such as phosphoramidate, or 
phgosphorothioate linkages in the phosphate backbone of the nucleotide 
sequences, may also provide resistance to nuclease attack. Binding 
affinity may also be optimised in particular circumstances, by providing 
nucleotides solely in the form of ribonucleotides, deoxyribonucleotides, 
or combinations thereof. In some circumstances it may be necessary to 
optimise the composition of the groups X and Y, to maximize target RNA 
cleavage. The cleavage activity of endonucleases having flanking 
nucleotide sequences which hybridise to target sequences and which are 
comprised wholly of deoxyribonucleotides may, in some circumstances, have 
reduced activity. In such circumstances optimisation may involve providing 
a mixture of deoxyribonucleotides and ribonucleotides in the nucleotide 
sequences X and Y. For example, nucleotides in the endonuclease which are 
proximal to the cleavage site in a target RNA may be in the form of 
ribonucleotides. The nucleotides A.sup.11 and N.sup.12 of the formula (II) 
interact with the target sequence adjacent to the cleavage site, with 
A.sup.11 interacting with the U of the target sequence X'UY', where X' and 
Y' are as herein defined. The nucleotide N.sup.12 is selected to be 
complementary to the nucleotide represented by X'. These nucleotides, or 
nucleotides in a 5' direction may, for example, be in the form of 
ribonucleotides. Where a target sequence is shown to be relatively 
resistant to certain embodiments of endonucleases of this invention, it 
may be necessary to provide nucleotide sequences X and Y partly or wholly 
in the form of ribonucleotides. Where desired, protection from nuclease 
attack as will be hereinafter described. 
The respective 5' and 3' termini of the groups X and Y may be modified to 
stabilise the endonuclease from degradation. For example, blocking groups 
may be added to prevent terminal nuclease attack, in particular 3'-5' 
progressive exonuclease activity. By way of example, blocking groups may 
be selected from optionally substituted alkyl, optionally substituted 
phenyl, optionally substituted alkanoyl. Optional substituents may be 
selected from C.sub.1 -C.sub.5 alkyl; halogen such as F, Cl or Br; 
hydroxy; amino; C.sub.1 -C.sub.5 alkoxy and the like. Alternatively, 
nucleotide analogues such as phosphothioates, methylphosphonates or 
phosphoramidates or nucleoside derivatives (such as .alpha.-anomers of the 
ribose moiety) which are resistant to nuclease attack may be employed as 
terminal blocking groups. 
Alternatively, groups which alter the susceptibility of the endonuclease 
molecule to other nucleases may be inserted into the 3' and/or 5' end of 
the endonuclease. For example, 9-amino-acridine attached to the 
endonuclease may act as a terminal blocking group to generate resistance 
to nuclease attack on the endonuclease molecules and/or as an 
intercalating agent to aid endonucleolytic activity. It will be readily 
appreciated that a variety of other chemical groups, e.g. spermine or 
spermidine could be used in a related manner. 
Endonucleases of this invention may be covalently or non-covalently 
associated with affinity agents such as proteins, steroids, hormones, 
lipids, nucleic acid sequences, intercalating molecules (such as acridine 
derivatives, for example 9-amino acridine) or the like to modify binding 
affinity for a substrate nucleotide sequence or increase affinity for 
target cells, or localisation in cellular compartments or the like. For 
example, the endonucleases of the present invention may be associated with 
RNA binding peptides or proteins which may assist in bringing the 
endonuclease into juxtaposition with a target nucleic acid such that 
hybridization and cleavage of the target sequence may take place. 
Nucleotide sequences may be added to the 5' and 3' ends of the groups X 
and Y to increase affinity for substrates. Such additional nucleotide 
sequences may form triple helices with target sequences (Strobel, S.A., et 
al., (1991) Nature 350: 172-174 and references therein which are 
incorporated by reference) which may enable interaction with 
intramolecularly folded substrate. Alternatively, modified bases 
(non-natural or modified bases as described in Principles of Nucleic Acid 
Structure, Supra) bases within the additional nucleotide sequences may be 
used that will associate with either single stranded or duplex DNA 
generating base pair, triplet, or quadruplet, interactions with 
nucleotides in the substrate. Suitable bases would include inosine, 
5'-methylcytosine, 5'-bromouracil and other such bases as are well known 
in the art, as described, for example, in Principles of Nucleic Acid 
Structure, Supra. 
In accordance with another aspect of this invention there is provided an 
endonuclease of the formula (I) as hereinbefore described wherein one of 
said nucleotide sequences X or Y includes the target polynucleotide which 
is cleaved by the endonuclease. Such an embodiment may cause the release 
of active RNA fragments on cleavage of a target RNA, which fragments may 
themselves possess endonuclease activity. 
In accordance with another aspect of this invention, there is provided a 
poly-endonuclease of the formula (IV): 
EQU Y--M--Y--(X--M--Y).sub.n 
where 
X, Y and M are as previously defined, and 
n is an integer of from 1 to 100. 
Poly-endonucleases have the potential to act as anti-sense molecules 
(Helene, C. and J-J Toulme (1990) Biochemica, Biophysica, Acta 1049: 
99-125) as well as endonucleases. By "antisense" is meant the formation of 
a duplex or double stranded sequence as a result of base pairing between 
complementary bases of a target sequence and an antisense oligonucleotide, 
which prevents translation of said sequence as a result of duplex 
formation or the creation of a template for cleavage of the RNA by RNase 
H, a cellular ribonuclease which acts to cleave the RNA component of 
hybridised RNA and DNA sequences. The possibility of acting as antisense 
may also arise when the groups X and Y of the endonuclease of this 
invention contain a significant number of nucleotides, such as 30 or more 
nucleotides. A duplex formed between such an endonuclease and a target 
sequence may not dissociate or readily melt under ambient conditions. 
In accordance with yet another aspect of this invention, there is provided 
a composition comprising an effective amount of an endonuclease of the 
formula (I) as hereinbefore defined either alone or in association with 
one or more pharmaceutically, veterinarally or agriculturally acceptable 
carriers or excipients. 
An effective amount or a therapeutically effective amount of an 
endonuclease of the formula (I) is an amount effective to cause cleavage 
of target RNA and/or inactivation thereof (such as by the provision of an 
endonuclease of the formula (I) wherein the nucleotide sequences X and Y 
comprise a significant number of nucleotides, such that the endonuclease 
essentially binds irreversibly to the target) so as to ameliorate disease 
in a human, animal or plant subject. What constitutes an effective amount 
of an endonuclease will vary depending on the nature of the disease being 
treated, the mode of application of the endonuclease to the subject, 
health of the subject, weight of the subject, and other like factors, as 
are well known in the pharmaceutical art to be associated with 
pharmaceutical effectiveness (see The Pharmacological Basis of 
Therapeutics, 4th Edition, Lewis S. Goodman and Alfred Gilman, 1970, The 
Macmillan Company). What constitutes a therapeutically effective dose in 
any particular circumstance can be readily determined according to 
standard procedures known in the pharmaceutical art (see The 
Pharmacological Basis of Therapeutics, Supra). For example, titration 
experiments may be employed where the effect of an endonuclease in tissue 
culture is studied to determine toxicity and effectiveness. Thereafter, 
trials may be conducted on animals and thereafter human patients to 
determine toxicity, effectiveness, preferred mode of administration and 
the like. Such investigations are routine in the pharmaceutical art as 
mentioned above, and thus a therapeutically effective amount of an 
endonuclease may be readily determined without undue experimentation for 
medical or veterinary purposes. 
A therapeutically effective amount of an endonuclease of the present 
invention would generally comprise from about 1 nM to about 1 mM 
concentration in a dosage form, such as a cream for topical application, a 
sterile injectable composition, or other composition for parenteral 
administration. In respect of topical formulations, it is generally 
preferred that between about 50 .mu.M to about 500 .mu.M endonuclease be 
employed. Endonucleases comprising nucleotide derivatives, which 
derivatives may involve chemically modified groups, such as 
phosphorothioate or methyl phosphonate derivatives may be active in 
nanomolar concentrations. Such concentrations may also be employed to 
avoid toxicity. 
Therapeutic strategies involving treatment of disease employing 
endoribonucleases of this invention are generally the same as those 
involved with antisense approaches, such as described in the anti-sense 
bibliography of Chrisley, L.A. (1991) Antisense Research and Development, 
1: 65-113, which reference and all references therein are incorporated by 
reference. Particularly, concentrations of endonucleases utilised, methods 
and modes of administrations, and formulations involved may be the same as 
those employed for antisense applications. 
By way of example only, therapeutic compositions of this invention may be 
directed against Herpes Simplex virus types 1 and 2, psoriasis, cervical 
preneoplasia, papilloma disease, and bacterial and prokaryotic infection. 
Such treatments may, for example, involve topical application of 
endonuclease to the site of disease. For example, in the treatment of 
Herpes virus lesions, endonucleases may be formulated into a cream 
containing a concentration of 1 nM to 1 mM endonuclease. The cream may 
then be applied to the site of infection over a 1 to 14 day period in 
order to cause amelioration of symptoms of the infection. Prior to the 
final development of topical formulations for the treatment of Herpes 
virus infection, effectiveness and toxicity of the endonucleases and 
formulations involving them may, for example, be tested on an animal 
model, such as scarified mouse ear, to which virus particles, such as 
2.times.10.sup.6 plaque forming units are added. A titre of infectious 
virus particles in the ear after treatment can then be determined to 
investigate effectiveness of treatment, amount of nuclease required and 
like considerations. 
Similar investigations in animal models prior to human trialing may also be 
conducted, for example, in respect of the treatment of psoriasis, 
papilloma disease, cervical preneoplasia, and in diseases such as HIV 
infection, bacterial or prokayrotic infection, viral infection and various 
neoplastic conditions, which neoplastic conditions involve a deleterious 
RNA species. 
Pharmaceutically and veterinarally acceptable carriers and excipients are 
well known in the art, and include carriers such as water, saline, 
dextrose and various sugar solutions, fatty acids, liposomes, oils, skin 
penetrating agents, gel forming agents and the like, as described for 
example in Remington's Pharmaceutical Sciences, 17th Edition, Mack 
Publishing Co., Easton, Pa., Edited by Ostol et al., which is incorporated 
herein by reference. 
Compositions for topical application are generally in the form of creams, 
where the endonucleases of this invention may be mixed with viscous 
components. In such embodiments, the endonucleases of this invention may 
be incorporated into liposomes or other barrier type preparations to 
shield the endonucleases from nuclease attack or other degradative agents 
(such as endonucleases and adverse environmental conditions such as UV 
light). 
Compositions may be provided as unit dosages, such as capsules (for example 
gelatin capsules), tablets, suppositories and the like. Injectable 
compositions may be in the form of sterile solutions of endonuclease in 
saline, dextrose or other media. Compositions for oral administration may 
be in the form of suspensions, solutions, syrups, capsules, tablets and 
the like. Endonucleases may also be provided in the form of sustained 
release articles, impregnated bandages, patches and the like. 
Pharmaceutical compositions which may be used in this invention are 
described, for example, in Remington's Pharmaceutical Sciences, Supra. 
The endonucleases of this invention may provided in a composition with one 
or more anti-viral, anti-fungal, anti-bacterial, anti-parasitic, 
anti-protazoan or anthelmentic agents, herbicides, pesticides or the like, 
for example as described in the Merck Index (1989) 11th Edition, Merck & 
Co. Inc. 
Agriculturally acceptable carriers and excipients are well known in the art 
and include water; surfactants; detergents; particularly biodegradable 
detergents; talc; inorganic and/or organic nutrient solutions; mineral 
earths and clays; calcium carbonate; gypsum; calcium sulfate; fertilisers 
such as ammonium sulfate, ammonium phosphate and urea; and natural 
products of vegetable origin such as, for example, grain, meals and 
flours, bark meals; and the like. 
The endonucleases of this invention have extensive application, stemming 
from the fact that virtually any RNA sequence may be cleaved by the 
endonucleases. The target nucleotide sequence GUC occurs, on a random 
basis, approximately once every 64 bases. The general endonuclease 
cleavage site X'UY', wherein X' and Y' are any nucleotide, occurs wherever 
the base uracil is present in an RNA, and thus any target RNA should be 
cleavable, albeit at different efficiencies, using the endonucleases of 
this invention. 
For in-vitro use, the endonucleases of this invention are generally reacted 
with a target RNA which contains one or more suitable cleavage sites. 
Optionally, the target RNA may be purified or substantially purified. The 
nucleotide sequences X and Y of the endonucleases of this invention are 
selected so as to specifically hybridise or form a double-stranded DNA 
duplex with a target RNA whereafter cleavage takes place. Accordingly, 
target RNA may be specifically cleaved in-vitro in the presence of other 
RNAs which themselves would not be cleaved by the endonucleases of this 
invention. 
The endonucleases may be utilised in a manner similar to restriction 
endonucleases, that is for the specific cleavage of RNA to facilitate RNA 
manipulation. All that is required for such manipulations is that the 
target RNA to be cleaved contains a uracil base and thus a suitable 
cleavage site. 
Endonucleases of this invention may be utilised in diagnostic procedures, 
such as the mapping or fingerprinting of RNA. Specifically, the 
endonucleases of this invention would enable mapping of RNA and may be 
used to detect mutations in RNA sequence. Such procedures may be used in 
research and may also have forensic and other diagnostic applications. 
RNA cleavage products in-vitro may be readily detected, for example, by 
visualisation on acrylamide or agarose gels where the amounts of RNA 
cleaved are sufficiently large for direct visualisation after separation 
and reaction with nucleotide visualisation agents, such as ethidium 
bromide. Alternatively, where the target RNA cleaved is present in small 
amounts, such as in a sample containing many RNAs, cleavage products may, 
for example, be detected by using radiolabelled probes of sequence 
complementary to the target sequence, or amplification techniques such as 
PCR (Sambrook et al., Supra). 
A target RNA for cleavage in-vitro may be derived from any source, and may 
be of animal, viral, bacterial, plant, synthetic, or other origin. As RNA 
is common to all known living organisms, this invention may be utilised to 
cleave any RNA species having a suitable cleavage site as mentioned 
previously. 
In-vitro cleavage of a target RNA is simply carried out by reacting the 
target RNA whether in purified, semipurified or unpurified form, or a 
sample containing the target RNA, with an effective amount of an 
endonuclease under reaction conditions facilitating RNA cleavage. Suitable 
reaction conditions include a reaction temperature of about 4.degree. C. 
to about 60.degree. C. (preferably about 20.degree. to 55.degree. C.), pH 
from about 7.0 to about 9.0, and Mg.sup.2+ from about 1 nM to about 100 mM 
(preferably 1 to 20 mM). The endonuclease may be present in an equimolar 
ratio to the substrate, or in excess thereof. As the endonucleases of this 
invention may act as enzymes, with each endonuclease cleaving multiple 
target sequences, the endonuclease may be provided in less than an 
equimolar ratio to target RNA. 
According to an aspect of this invention, there is provided a method for 
the cleavage of the target nucleotide sequence in-vitro which comprises 
reacting said target nucleotide sequence or a sample containing said 
target nucleotide sequence with an endonuclease as described herein 
wherein nucleotide sequences X and Y of the nuclease are selected so as to 
be complementary to nucleotide sequences flanking a selected cleavage site 
of the target RNA, such that on hybridisation of the endonuclease to the 
target RNA, said target RNA is cleaved at the selected cleavage site. 
In circumstances where the nucleotide sequences X and Y comprise a 
significant number of nucleotides, such as 30 or more nucleotides, the 
duplex formed on reaction of the endonuclease with its complimentary 
target may not readily dissociate and hence such target RNAs may be 
inactivated not only by cleavage, but by blocking translation into a 
desired protein product, RNase H digestion, and/or prevention of 
interaction with other RNAs. 
The endonucleases of this invention may be used for RNA cleavage in-vivo 
both in prokaryotic and eukaryotic cells. 
The endonucleases of this invention may be utilised to cleave any RNA 
within a cell which contains the cleavage site X'UY' as described herein. 
Virtually all cellular RNAs would therefore be targetable utilising 
endonucleases of this invention. 
Cleavage of target RNA within cells, such as bacterial cells, yeast cells, 
or animal cells, or the cleavage of a target RNA within the cells of an 
organism, such as a plant or animal, may result in phenotypic 
modifications or the treatment of disease or infection. 
Phenotypic changes in plant cells or plants may include drought resistance, 
salinity resistance, resistance to fungal, viral or bacterial infection; 
modifications of growth characteristics; sterility; fruit production; 
flowering; senescence and the like. It is evident that once one or more 
RNAs involved in determining phenotype are identified, such RNAs may be 
inactivated by cleavage utilising the endonucleases of this invention and 
thus the phenotype of the plant or plant cell altered. 
Phenotypic modifications within animals (including in some applications 
man) which may be effected by cleaving and thus inactivating target RNAs 
associated with phenotype would include growth characteristics of animals, 
fertility, skin/cosmetic modifications, reproductive characteristics, 
disease resistance and the like. Myriad applications arise for phenotypic 
modifications in animals, and plants as previously mentioned. Once one or 
more RNAs associated with a given phenotype are identified and their 
sequence determined, endonucleases may be targeted against such RNAs for 
their inactivation with consequential phenotypic modification. 
Prokaryotic or eukaryotic cell cultures may be phenotypically modified by 
treatment with endonucleases of this invention. For example, bacterial 
cultures or yeast cultures involved in production of food components (such 
as cheese, bread and dairy products) and alcoholic beverage production may 
be treated so as to modify enzyme content, flavour production, cell growth 
rate, culture conditions and the like. 
The endonucleases of this invention may also be used to treat disease or 
infection in humans, animals, plants, or prokaryotic or eukaryotic cells. 
The ability to treat disease or infection is based on the fact that the 
endonucleases of this invention are capable of cleaving any RNA which 
contains a suitable cleavage site, such as defined by the generic cleavage 
site X'UY', where X' and Y' represent any nucleotide (preferably wherein 
the cleavage site is GUC) as described previously. Most RNAs will contain 
one or more suitable cleavage sites. 
The period of treatment would depend on the particular disease being 
treated and could be readily determined by a physician. Generally 
treatment would continue until the disease being treated was ameliorated. 
Examples of human and animal disease which may be treated with the 
endonucleases of this invention include Herpes Simplex Virus infection 
(such as targeting cleavage of early genes 4 and 5), psoriasis, cervical 
preneoplasia, papilloma disease, HIV infection (such as targeting the 
HIV-1 gag transcript and HIV-1 5'ltr splice site), bacterial and 
prokaryotic infection, viral infection and neoplastic conditions 
associated with the production of aberrant RNAs such as occurs in chronic 
myeloid leukemia. Diseases or infections which may be treated in plants 
with endonucleases of this invention include fungal infection, bacterial 
infections (such as Crown-Gall disease) and disease associated with plant 
viral infection. 
Eukaryotic and prokaryotic cells in culture may, for example be protected 
from infection or disease associated with mycoplasma infection, phage 
infection, fungal infection and the like. 
For the in-vivo applications of the endonucleases of this invention in 
humans, animals, plants, and eukaryotic and prokaryotic cells, such as in 
phenotypic modification and the treatment of disease, it is necessary to 
introduce the endonuclease into cells whereafter, cleavage of target RNAs 
takes place. 
Methods for the introduction of RNA and DNA sequences into cells, and the 
expression of the same in prokaryotic and eukaryotic cells are well known 
in the art for example as discussed in Cotten, M. (1990) Tibtech 8: 
174-178; and Friedman, T. (1989) Science 244: 1275-1280 (both of which 
references are incorporated herein by reference. The same widely known 
methods may be utilised in the present invention. 
The endonucleases of this invention may be incorporated into cells by 
direct cellular uptake, where the endonucleases of this invention would 
cross the cell membrane or cell wall from the extracellular environment. 
Agents may be employed to enhance cellular uptake, such as liposomes or 
lipophilic vehicle, cell permeability agents, such as dimethylsulfoxide, 
and the like. 
Endonucleases of this invention may be incorporated and expressed in cells 
as a part of a DNA or RNA transfer vector, or a combination thereof, for 
the maintenance, replication and transcription of the endonuclease 
sequences of this invention. 
Transfer vectors expressing endoribonucleases of this invention may be 
capable of replication in a host cell for stable expression of 
endonuclease sequences. Alternatively, transfer vectors encoding 
endonuclease sequences of this invention may be incapable of replication 
in host cells, and thus may result in transient expression of endonuclease 
sequences. Methods for the production of DNA and RNA transfer vectors, 
such as plasmids and viral constructs are well known in the art and are 
described for example by Sambrook et al. (Supra). 
Transfer vectors would generally comprise the nucleotide sequence encoding 
the endonuclease of this invention, operably linked to a promoter and 
other regulatory sequences required for expression and optionally 
replication in prokaryotic and/or eukaryotic cells. Suitable promoters and 
regulatory sequences for transfer vector maintenance and expression in 
plant, animal, bacterial, and other cell types are well known in the art 
and are described for example in Hogan, B. et al., (1986) Manipulating the 
Mouse Embryo, A Laboratory Manual, Cold Spring Harbor; and Science (1989) 
244: 1275-137, which are incorporated herein by reference. 
Transfer vectors or nucleic acid sequences encoding or comprising the 
endonucleases of this invention may be incorporated into host cells, such 
as plant or animal cells, by methods well known in the art (for example, 
as described by Cotten and Friedman (Supra), such as microinjection, 
electroporation, receptor-mediated endocytosis, transformation of 
competent cells such as protoplasts or bacterial cells treated with metal 
ions such as calcium chloride, cationic or other liposomes, viral or 
pseudovirus vectors, DEAE-Dextran, or by using projectiles to penetrate 
cell walls and thereby deliver the desired nucleic acid sequence. 
In accordance with a still further aspect of this invention, there is 
provided a transfer vector which encodes a nucleotide sequences encoding 
an endonuclease as described herein. 
Nucleotide sequences encoding the endonucleases of this invention may be 
integrated into the genome of a eukaryotic or prokaryotic host cell for 
subsequent expression (for example as described by Sambrook et al., 
Supra). Genomic integration may be facilitated by transfer vectors which 
integrate into the host genome. Such vectors may include nucleotide 
sequences, for example of viral or regulatory origin, which facilitate 
genomic integration. Methods for the insertion of nucleotide sequences 
into a host genome are described for example in Sambrook et al. and Hogan 
et al., Supra. 
Genomically integrated nucleic acid sequences encoding the endonucleases of 
this invention generally comprise a promoter operably linked to the 
nucleotide sequence encoding the endonuclease of this invention, and 
capable of expressing said endonuclease in a eukaryotic (such as animal or 
plant cells) or prokaryotic (such as bacteria) host cells. 
Endonucleases of this invention may be involved in gene therapy techniques, 
where, for example, cells from a human suffering from a disease, such as 
HIV are removed from a patient, treated with the endonuclease to 
inactivate the infectious agent, and then returned to the patient to 
repopulate a target site with resistant cells. In the case of HIV, 
nucleotide sequences encoding endonucleases of this invention capable of 
inactivating the HIV virus may be integrated into the genome of 
lymphocytes or be present in the cells a transfer vector capable of 
expressing endonucleases of this invention. Such cells would be resistant 
to HIV infection and the progeny thereof would also confer such 
resistance. 
In accordance with an aspect of this invention, there is provided a method 
for the cleavage of a target nucleic acid sequence either in-vivo or 
in-vitro which comprises reacting a target nucleotide sequence with an 
effective amount of an endonuclease as described herein, said endonuclease 
being capable of effecting specific cleavage of said target at a site 
selected such as to cleave and inactivate the target nucleic acid 
sequence. 
In accordance with another aspect of this invention, there is provided a 
method for the treatment of disease or infection in a human, animal, 
plant, or prokaryotic or eukaryotic cell, which is associated with the 
presence of a deleterious RNA, which method comprises treating said human, 
animal, plant, prokaryotic or eukaryotic cell with an effective amount of 
an endonuclease as described herein, either alone or in association with a 
pharmaceutically, veterinarally, or agriculturally acceptable carrier or 
excipient, which endonuclease is capable of cleaving and thus inactivating 
said deleterious RNA. 
Recombinant DNA manipulations referred to above are well known in the art, 
and are described for example by Sambrook et al., Supra. 
In another aspect of this invention, there is provided an animal or plant 
which comprises one or more cells which have been modified by, or contain, 
encode and/or express an endonuclease as herein defined. 
The endonucleases of this invention may be produced by nucleotide synthetic 
techniques which are well known in the art, and described for example by 
Carruthers et al. (Methods in Enzymology (1987) 154: 287-313), Foehler et 
al. (Nucleic Acids Research (1986) 14: 5399-407) and Sprat et al. 
(Oligonucleotide Synthesis--A Practical Approach, IRL Press, Oxford (1984) 
M. J. Gait--Editor, pp. 83-115), all of which are incorporated herein by 
reference. Generally, such synthetic procedures involve the sequential 
coupling of activated and protected nucleotide bases to give a protected 
nucleotide chain, whereafter protecting groups may be removed by suitable 
treatment. Alternatively, the endonucleases in accordance with this 
invention may be produced by transcription of nucleotide sequences 
encoding said endonucleases in host-cells or in cell free systems 
utilizing enzymes such as T3, SP6 or T7 RNA-polymerase (Sambrook et al., 
Supra). 
The catalytic region M of the endonucleases of this invention is of a 
reduced size compared with what may have been considered necessary from 
knowledge in the prior art. The absence of a conventional base-paired stem 
structure provided by an embodiment of the endonucleases of this invention 
may reduce steric interference of the endonuclease with its substrate or 
other nucleic acid structures and may also reduce the likelihood of 
non-favourable base pairing interactions particularly in the in-vivo 
context when the endonuclease may be in association with a large number of 
nucleic acids, in addition to the specific target which it is engineered 
to cleave. 
The inclusion of deoxyribonucleotides in the endonuclease structure in 
certain embodiments of this invention may provide protection against 
ribonuclease degradation. Also, endonucleases comprised of RNA/DNA may not 
provide a substrate for unwinding/modifying enzymes which compromises some 
anti-sense applications. The reduced size of various embodiments of the 
endonuclease of this invention when compared with other endonucleases 
known in the art may serve to improve the economics of synthesis of the 
endonuclease and may also serve to improve the ease and efficiency of the 
introduction of the endonuclease into host ecells. 
Poly-endonucleases as described herein may be designed to have cleavage 
sequences within the flanking regions X and Y which link catalytic 
domains. Such poly-endonucleases may autocatalytically liberate multiple 
individual endonucleases in cells, thus increasing the local concentration 
of endonucleases. 
The oligonucleotide backbone (that is, phosphodiester linkages) of 
compounds of the formula (I) may be modified in a variety of ways, for 
example, in the same manner as for DNA antisense oligonucleotides. 
Methylphosphonate, phosphorothioate and phosphoramidate linkages may be 
used to replace conventional phosphodiester linkages. Additionally, 
ribonucleotides may be substituted with modified nucleotides and/or bases, 
for example, 2'methoxyribonucleotides or [.alpha.]-anomers as described 
herein. These modifications may confer nuclease resistance and improve 
biological halflife and/or cellular uptake of endoribonucleases. 
Phosphorothioate linkages confer RNAse H sensitivity to the RNA component 
of RNA/DNA duplexes. Methylphosphonate linkages confer RNase H resistance 
to this same component. 
Various embodiments to the present invention will now be described, by way 
of non-limiting example only, in the following examples. 
EXAMPLE 1 
Endonuclease Synthesis 
All endonucleotides were synthesized or either or both an Applied 
Biosystems 380 or 391 synthesizer using 2-cyanoethylphosphoramidite 
chemistry. DNA monomers and RNA monomers, protected at the 2' position 
with a t-butyldimethylsilyl group, were obtained from commercial 
suppliers. All oligonucleotides, with 5'-trityl groups removed, were 
worked up as follows: the oligonucleotide was cleaved from the column in 
3:1 NH.sub.4 OH/ethanol, and heated overnight at 55.degree. C. The 
solutions were evaporated to near dryness, taken up in H.sub.2 O/ethanol 
3:1 and dried, and repeated. The amount of material was estimated at this 
stage by measuring the UV absorbance. The 2' group was deprotected by 
treatment overnight with 1M tetrabutylammonium fluoride in THF (10 .mu.L 
per OD.sub.260). The tetrabutylammonium ions were removed by passage twice 
through a Dowex 50X8-200 (trade mark) cation-exchange column in the 
Na.sup.+ form, the volume of the eluate was reduced with 2-butanol, and 
the oligonucleotide precipitated with sodium acetate and ethanol. The 
oligonucleotide was then purified by electrophoresis on a 10-20% 
(depending on length) acrylamide gel containing 7M urea. The band of 
interest was visualised by UV shadowing or ethidium bromide staining, 
excised and soaked in water. The oligonucleotide solution was removed from 
the gel slices, concentrated with 2-butanol, washed with phenol/chloroform 
and ether. The oligonucleotide was then precipitated with sodium acetate 
and ethanol, washed with cold 80% ethanol, redissolved in 10 mM Tris-Cl, 
pH 8.0, 2 mM EDTA, quantified by UV spectroscopy, and frozen. Purity of 
the oligonucleotides was determined by labelling the 5' end with .sup.32 p 
phosphate and running out in a denaturing gel. Olibonucleotides were 
phosphorylated using standard conditions, except that several units of 
pancreatic ribonuclease inhibitor were added to the reaction mixture. 
Concentration of labelled material was determined by pooling all waste 
from the phosphorylation procedure, drying down, and running on a gel 
alongside a known fraction of the labelled oligonucleotide; and bands were 
excised and counted, and from this the amount of material lost in the 
phosphorylation procedure was known, and the concentration of the labelled 
oligonucleotide determined. 
Reaction Conditions 
Typically reactions were conducted in 50 mM Tris.HCl, pH 8.0, 10 mM 
MgCl.sub.2 at 37.degree. C.; substrate concentration was 100 nM, and 
endonuclease concentration was 100 or 600 nM in all reactions. The 
reactions were conducted in a 30 .mu.L volume over a range of 30 minutes 
to 4 hours. The molar ratio of endonuclease to substrate was 1:1 or 6:1. 
The endonuclease and substrate (.sup.32 p labelled) were heated separately 
in reaction buffer to 70.degree. C. for three minutes, then snap-cooled 
before mixing and subsequent incubation. Departures from the standard 
reaction conditions were taken as needed in experiments aimed at 
determining the temperature/activity profile, magnesium dependency, and pH 
dependency and turnover of the endonuclease mediated cleavage reaction. 
Samples were then analyzed by electrophoresis in 15% acrylamide gels 
containing 7M urea as a denaturant. The substrate and product of cleavage 
were visualised by autoradiography, and gel slices corresponding to their 
positions were excised and quantified by Cerenkov counting. 
EXAMPLE 2 
Growth Hormone RNA Targeted Endonucleases 
Base sequence, schematic representation and names of various endonucleases 
are set out below. Conserved ribonucleotides are depicted by bold type and 
thick lines. Other ribonucleotides are depicted by upper case letters and 
thin lines. Deoxyribonucleotides are depicted by lower case letters and 
wavy lines. The nomenclature for the endonucleases is as follows: R 
indicates a ribozyme containing helix II, M denotes an endonuclease not 
containing helix II, and MS denotes a endonuclease and substrate in the 
same molecule; the numbers and nucleotide designations indicates the bases 
in the connector, and the RNA or DNA refers to the nucleotides in the arms 
that form helices I and III with the substrate. Double helices I, II and 
III are as described by Forster and Symons (Cell (1987), 49: 211-220). 
A further series of growth hormone RNA targeted endonucleases were 
synthesised based on the M4t,DNA construct and having different numbers 
and types of nucleotides in the connector. These are as follows: 
M2t,DNA 
M3t,DNA 
M5t,DNA 
Mttct,DNA 
MttPDt,DNA 
PD refers to 1,3-propanediol which was used in place of a nucleoside. 
An endonuclease R4U,DNA was also synthesized. This endonuclease is the same 
as R4U,RNA depicted above, except that the RNA flanking sequences are 
replaced with DNA. 
SUBSTRATES 
Rat Growth Hormone 21 Mer's 
All of the above endonucleases were reacted with a ribonucleotide sequence 
corresponding to a portion of the rat growth hormone gene and having the 
sequence: 
EQU GHS1 5' A C C U G C G G G U C* A U G A A G U G U C 3' 
A second substrate (GHS2) corresponding to GHS1 but where all nucleotides 
except C* were deoxyribonucleotides was also synthesized. Endonucleases 
M4U,RNA and M4U,DNA were reacted with this substrate. 
Kruppel RNA 
The endonuclease M4t,DNA, Kr1079 (comprising 34 nucleotides and having 
flanking sequences of DNA designed to hybridise to the Kruppel target RNA) 
was tested against a short synthetic RNA substrate of 21 nucleotides and a 
RNA substrate of approximately 1.9 Kb, both containing the same cleavage 
site. 
The Kr RNA transcript was prepared by inserting cDNA encoding the Kr 
transcript into a plasmid containing the T7RNA polymerase promoter. The Kr 
transcript was then transcribed with T7-polymerase. 
The synthetic 21 mer was chemically synthesised and contained the same 
cleavage site as the longer RNA transcript. The 21 mer comprised the 
following sequence: 
EQU A U U U G C G A G U C* C A C A C U G G A G 
where C* is a ribonucleotide. 
In-vivo testing of activity of an anti-Kruppel endonuclease could be 
accomplished by microinjection of Drosophila embryos prior to the stage of 
syncytial blastoderm, in order to inactivate the 2.3 Kb RNA Kruppel 
transcript. Embryos (cuticlised embryos) can be assayed for abberant 
segmental pattern one to two days after egg laying. 
Platelet Derived Growth Factor (PDGF) 
The endonuclease Mttct, DNA, PDGF (a 30 mer, having flanking DNA sequences 
designed to hybridise to the PDGF target RNA) was reacted with a 666 base 
RNA transcript corresponding to exons 2 and 6 of the PDGF A gene from 
humans. A 666 base pair RNA transcript was transcribed in-vitro from a 
PDGF gene fragment using T3 polymerase. 
RESULTS 
A. All growth hormone RNA targetted endonucleases cleaved GHS1 with varying 
activities. In keeping with the endoribonuclease of Haseloff and Gerlach 
(Supra) and those of other investigators, no endonuclease cleaved 100% of 
product over the reaction period. Endonucleases M4T,DNA; M4U,DNA; M4A,DNA; 
M4U,RNA; M3T,DNA; M5T,DNA; and MTTCT,DNA; all cleaved between about 50-60% 
of substrate over the reaction period, this being about the same as the 
control R4U,RNA. Endonucleases containing less than four nucleotides in 
the connector (corresponding to the group P of the formula (II)) were 
somewhat less active in the test assay. 
Experiments were conducted to examine the enzymatic turn over, temperature 
activity profile, magnesium dependency and pH dependence of representative 
minizyme M4T,DNA and a reference control, R4U,RNA, corresponding to the 
endoribonuclease of Haseloff and Gerlach (Supra). These experiments showed 
that both endonucleases behaved as enzymes exhibiting turn over, achieving 
cleavage of target substrate at pH 7.5, 37.degree. C. and greater than or 
equal to 1 mM Mg2+. 
It is clear from these results that functional endonucleases capable of 
cleaving target substrates may contain hybridising arms comprised of DNA 
or RNA, combinations thereof (corresponding to the groups X and Y of 
formula (I)); a connector comprised of a non-base paired RNA or DNA 
sequence, or a partly base paired RNA sequence and that the number and 
nature of nucleotides in the connecting sequence is not of importance. 
The endonuclease MttPDt,DNA was active and shows that nucleotides may be 
replaced with chemical linking groups without loss of endonuclease 
activity. On the basis of this experiment, it is apparent that nucleotides 
in the connector sequence (corresponding to the group P of the formula 
(II)) may be replaced with linker sequences. 
B. The substrate GHS2 was cleaved by M4U,RNA but not M4U,DNA. It is 
presently unclear why the M4U,DNA endonuclease was not active against this 
substrate although this was obviously suggestive that the structure of a 
DNA-DNA duplex is sufficiently different from that of an RNA-RNA or an 
RNA-DNA duplex of the same sequence so as to possibly alter the structure 
of the active site and render the endonuclease inactive. 
As the endonuclease M4U,RNA cuts a substrate made entirely of DNA, except 
for the central ribonucleotide, it is apparent that the only 2' hydroxyl 
on the substrate required for the cleavage reaction is on the nucleotide 
to be cleaved. 
By altering the nucleotide composition of the flanking sequences of the 
endonuclease (corresponding to the groups X and Y of the formula (II)), 
any nucleotide substrate containing a suitable cleavage site may be 
cleaved. Optimisation of the flanking nucleotides as hereinbefore defined 
may be required to facilitate cleavage, as illustrated by the fact that 
M4U,RNA cleaves a substrate made entirely of DNA except for the central 
ribonucleotide having the base C, whereas the endonuclease M4U,DNA does 
not cleave this substrate. 
Cleavage of the 1.9 Kb base Kruppel RNA and synthetic 21 mer RNA target 
hybrid was observed with the M4t,DNA,Kr1079 endonuclease. 
Cleavage of the 1.9 Kb Kruppel RNA transcript indicates that large RNAs can 
be selectively cleaved at a desired cleavage site. 
C. Similarly, an individual endonuclease effected efficient and specific 
cleavage of the long PDGF RNA transcript. 
A synthetic RNA linked in cis to its target sequence, MS4U,RNA, was also 
active in effective specific cleavage at the predicted target sequence, at 
nanomolar concentrations with a rate independent of concentration. This 
provides evidence that this endonuclease does not form polymers or high 
molecular weight structures to effect cleavage, but rather that an 
individual discrete nuclease is capable of effecting catalytic cleavage of 
a target nucleotide sequence. 
It is clear from the above examples that the endonucleases of this 
invention may be selectively used for the cleavage of any target RNA which 
contains an appropriate cleavage site (such as GUC) and whose nucleotide 
sequence is known. Accordingly, the endonucleases of this invention have 
wide application in the selective inactivation of target RNAs in-vivo and 
in-vitro and as such may be used for example for the treatment of disease 
in humans and animals, phenotypic alteration in animals and plants and 
other myriad applications.