Human papilloma virus inhibition by a hairpin ribozyme

Synthetic catalytic RNAs, i.e. ribozyme, including a hairpin portion, binding sites for binding to a human papilloma virus after viral base 419 and 434, respectively, and cleavage sites for cleaving the virus at the binding sites have been constructed.

TECHNICAL FIELD 
The present invention relates to an RNA catalyst, i.e. ribozyme, which 
cleaves Human Papilloma virus into a fragment having a 5' hydroxyl and a 
fragment having a 2',3' cyclic phosphate. The products of the reaction 
described herein resemble those resulting from the natural hydrolysis of 
RNA. 
BACKGROUND OF THE INVENTION 
Pappillomaviruses are small DNA viruses that induce the hyperproliferation 
of epithelial cells. Approximately 70 different genotypes have been 
isolated from humans. Some types (1, 2, 4, and 7) are associated with 
benign squamous papillomas (warts; condylomas) in humans, while at least 
two types (16 and 18) have been associated with human neoplastic and 
preneoplastic lesions..sup.7 
In the United States, cervical cancer affects approximately 8.6 women per 
100,000 each year. In woman, HPV-16 is frequently associated with latent 
infections, benign and premalignant cervical lesions (dysplasias/CIN) and 
half of invasive cervical carcinomas. In males, HPV-16 is associated with 
subclinical macular or clinical papular lesions. Bowenoid papulosis of the 
penis resembles carcinoma in situ. Cervical cancer, which kills at least 
500,000 women worldwide each year, proceeds through progressive cellular 
changes from benign condylomata to high-grade dysplasias/CIN before 
developing into an invasive cancer. Over five billion health care dollars 
are spent in the United States each year on the detection and treatment of 
these lesions. 
Epidemiology evidence indicates that up to 90% of all human and oral tumors 
harbor types of HPV that are able to immortalize primary human 
keratinocytes and transform rodent cells. The oncogene potential of HPV 
appears to be associated with products from two viral genes, E6 and E7. 
These products are required for the acquisition and maintenance of a 
transformed phenotype. The proteins encoded by these genes bind, with high 
affinity in neoplastic-associated types, to and neutralize the products of 
the Rb and p53 tumor suppressor cells..sup.15,20,21,22,23,24 
The current policy in genitourinary clinics is surgery for high-grade 
lesions due to the lack of superior alternatives. Cervical laser ablation 
therapy does not in the long term influence the natural history of 
cervical human papillomavirus-associated diseases in women. Interferons, 
per se, have been disappointing insofar as acute viral infection is 
concerned, usually because treatment cannot be started in time. Therefore, 
it has been assumed that any benefit with interferons is due to 
anti-proliferative effect and not due to antiviral. 
Combination chemotherapy is also in use in cancer therapy, and cisplatin is 
one of the drugs of choice for cervical cancer, alone or in combination 
with other chemotherapy agents. However, the current success obtained with 
chemotherapy treatment is poor. The response rate for combination 
cisplatin and 5FU treatment in phase II studies in cervical cancer 
patients is only effective in 22% of the patients while the same 
combination produced an 88% response in squamous cell carcinoma of the 
head and neck. 
The use of cytotoxic agents for cancer therapy has limitations because of 
toxic side effects and the development of multiple drug resistance. 
Therefore, there has been a consideration of a shift to therapy which does 
not involve direct toxic reaction, but which can modify the growth of 
tumor cells. 
Current new therapeutic suggestions for treatment of HPV infections have 
centered on the use of antisense oligonucleotides to interrupt viral mRNA 
utilization..sup.7,23,24 However, antisense therapy is limited by 
stoichiometric considerations..sup.19 
Ribozymes are RNA molecules that possess RNA catalytic ability (see Cech et 
al., U.S. Pat. No. 4,987,071) that cleave a specific site in a target RNA. 
The number of RNA molecules that are cleaved by a ribozyme is greater than 
the number predicted by stochiochemistry..sup.11,26 This provides an 
advantage over the antisense technology. 
Antisense therapy has two disadvantages when compared to ribozymes: (1) by 
its nature, the antisense molecule is not catalytic; and (2) antisense 
molecules are normally longer than the ribozyme target recognition 
sequence. This increases the likelihood of antisense molecules having a 
deleterious effect on similar mRNA sequences found in the same gene 
family. 
Ribozymes have been designed on the "hammerhead" motif..sup.10 However, 
catalytic RNAs such as those that were designed based on the "hammerhead" 
model have several limitations which restrict their use in vitro and may 
forestall their use in vivo. For example, the temperature optimum for the 
reaction is 50.degree.-55.degree. C., which is well above physiological, 
and the kcat (turnover number) is only 0.5/min even at 55.degree. 
C..sup.13,26 In addition, the Km is 0.6 .mu.M,.sup.26 meaning that the 
reaction requires high concentrations of substrate which makes it 
difficult, if not impossible, for the catalytic RNA to cleave low levels 
of target RNA substrate such as would be encountered in vivo. 
A "hairpin" motif has been found to be more efficient than the "hammerhead" 
motif..sup.11,12 Further, hairpin ribozymes have been used to cleave 
targets on HIV..sup.16,28 However, ribozymes for one virus generally will 
not cleave other virus species. Not only do the ribozymes require specific 
target sequences for cleavage, they require modifications in the ribozyme 
structure itself to be able to efficiently cleave a specific target. 
Currently, there is no ribozyme that has been shown to cleave HPV RNA and 
no site has been identified in the HPV that is capable of cleavage by a 
ribozyme. 
SUMMARY OF THE INVENTION AND ADVANTAGES 
According to the present invention, synthetic catalytic RNAs, i.e. 
ribozymes, including a hairpin portion, a binding site for binding to a 
human papilloma virus either after viral base 434 or after base 419 and a 
cleavage site for cleaving the virus at the binding site have been 
constructed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
A hairpin ribozyme containing a tetraloop modification was designed, tested 
and shown to cleave a specific sequence in the primary transcript from 
human papilloma virus type 16. The cleavage sites immediately followed 
nucleotide 434 and 419, respectively, in the sequence of this virus. 
Optimization of the ribozyme was carried out showing that an 8 nt helix 1 
was optimal for the 434 site and that a 7 nt helix i was optimal for the 
419 site. The time course of the reaction showed nearly complete cleavage 
of the substrate. 
Kinetic parameters for the 434 site were measured using standard Michaelis 
enzyme kinetic analysis. The Km for the reaction was 21 nM which shows 
very tight binding of the ribozyme and substrate. The kcat or turnover 
number was 0.083 min.sup.-1 to give an overall catalytic efficiency 
(kcat/Km) of 4 .mu.M.sup.-1 min.sup.-1. 
Kinetic parameters for the 419 site were also measured using standard 
Michaelis enzyme kinetic analysis. The Km for the reaction was 98 nM and 
the kcat or turnover number was 0.18 min.sup.-1 to give an overall 
catalytic efficiency (kcat/Km) of 1.8 .mu.M.sup.-1 min.sup.-1. 
The optimized target sites are shown in FIG. 1. Cleavage occurred after 
base 434 and after base 419, respectively, and before the GUC sequence 
shown as indicated in the diagram. This entire target sequence is part of 
the primary transcript (SEQ ID No:1) for the E6 and E7 regions of 
HPV16..sup.15,21 The cap for this mRNA is on nt 97. A splice donor exists 
at nt 226, and two splice acceptors exist at nt 409 and nt 526. As a 
result, three different E6-E7 mRNAs can be produced: E6E7, E6(I)E7, and 
E6(II)E7. E6E7 is the result of the full-length E6E7 transcript, in which 
the splice donor at nt 226 is not utilized. In E6E7, translation 
termination of E6 occurs at nt 557. E6(I)E7, the major transcript, is the 
result of utilization of the splice donor at nt 226 and the splice 
acceptor at nt 409, and its E6(I) translation termination signal is at nt 
415. This gives a truncated E6 coding region and a full-length E7. 
E6(II)E7, the minor transcript, is the result of utilization of the splice 
donor at nt 226 and the splice acceptor at nt 526, and its E6(II) 
translation termination signal is at nt 541 to give a truncated E6 coding 
region and a full-length E7..sup.15 
An RNA catalyst (ribozyme) has been identified comprising an RNA sequence 
which can cleave, with great precision, HPV. The target sequences for 
cleavage by the ribozymes are present in the primary transcript E6E7, and 
E6(I)E7, the major transcript. Cleavage of these transcripts would have 
the effect of lowering the production of full-length E6 and E7 proteins, 
both of which appear to play a key role in keratinocyte 
transformation..sup.20 
The hairpin ribozyme.sup.12 designed to cleave after the 434 site in HPV is 
shown in FIG. 2 and is designated RHPV434. In the preferred embodiment, 
this hairpin ribozyme has the tetraloop modification as shown..sup.2 The 
GUU sequence of Loop 3 of the basic structure has been replaced by a 
tetraloop sequence GGAC (UUCG) GUCC which in the present invention has 
been shown to generate a very stable structure with high catalytic 
efficiency. In particular, the invention comprises certain synthetic RNA 
catalysts capable of cleaving an RNA substrate which contains the target 
sequences: 
430-ACUG U*GUC CUGAAGA-444 (SEQ ID NO:2) 
430-ACUG U*GUC CUGAAGAA-445 (SEQ ID NO:3) 
430-ACUG U*GUC CUGAAGAAA-446 (SEQ ID NO:4) 
The hairpin ribozyme designed to cleave after the 419 site in HPV is shown 
in FIG. 6 and is designated RHPV419. In the preferred embodiment, this 
hairpin ribozyme also has the tetraloop modification as shown. The GUU 
sequence of Loop 3 of the basic structure has been replaced by a tetraloop 
sequence GGAC (UUCG) GUCC which in the present invention has been shown to 
generate a very stable structure with high catalytic efficiency. In 
particular, the invention comprises certain synthetic RNA catalysts 
capable of cleaving an RNA substrate which contains the target sequences: 
415-UAAC U*GUC AAAAGC-428 (SEQ ID NO:7) 
415-UAAC U*GUC AAAAGCC-429 (SEQ ID NO:8) 
415-UAAC U*GUC AAAAGCCA-430 (SEQ ID NO:9) 
415-UAAC U*GUC AAAAGCCAC-431 (SEQ ID NO:10) 
"Synthetic RNA catalyst," as used herein, means a catalyst which is not a 
naturally-occurring RNA catalyst, although "synthetic catalysts" may be 
truncated or altered versions of naturally-occurring catalysts. "Synthetic 
catalyst" include catalysts synthesized in vitro and catalysts synthesized 
in vivo. In particular, "synthetic catalysts" can include catalysts 
produced by hosts transformed by a vector comprising a sequence coding for 
the catalyst. 
RNA of any length and type may be used as the substrate as long as it 
contains the target sequence represented by the formula 5'-F.sub.1 
-CS-F.sub.2 -3'. In this formula, CS is the cleavage sequence, i.e., a 
sequence of bases containing the site at which the catalyst cleaves the 
substrate. CS is a short sequence of bases which does not base pair with 
the ribozyme, and in the present invention CS preferably has the sequence 
5'-NGUC-3', wherein N is any base, and the substrate is cleaved by the 
ribozyme between N and G to produce a fragment having an OH at the 5' end 
and a fragment having a 2,'3' cyclic phosphate at the 3' end. 
CS is flanked by two short base sequences F.sub.1 and F.sub.2 which do base 
pair with the RNA catalyst. F.sub.1 is preferably at least 3 bases in 
length, most preferably 4 bases in length. F.sub.2 is also preferably at 
least three bases in length, most preferably 6 to 12 bases in length. 
Ribozymes, according to the present invention, also include a substrate 
binding portion and a "hairpin" portion. The substrate binding portion of 
the catalyst is represented by the following formula: 
EQU 3'F.sub.4 -L.sub.1 -F.sub.3 -5' 
wherein, 
F.sub.3 is a sequence of bases selected so that F.sub.3 is substantially 
base paired with F.sub.2 (Helix 1, FIGS. 2 and 6) when the catalyst is 
bound to the substrate; 
F.sub.4 is a sequence of bases selected so that F.sub.4 is substantially 
base paired with F.sub.1 when the catalyst is bound to the substrate 
(Helix 2, FIGS. 2 and 6); 
The sequences of F.sub.3 and F.sub.4 are selected so that each contains an 
adequate number of bases to achieve sufficient binding of the RNA 
substrate to the RNA catalyst so that cleavage of the substrate can take 
place; and 
L.sub.1 is a sequence of bases selected so that L.sub.1 does not base pair 
with CS when the catalyst is bound to the substrate (Loop 1, FIGS. 2 and 
6). 
As used herein, "substantially base paired" means that greater than 65% of 
the bases of the two RNA sequences in questions are base paired, and 
preferably greater than 75% of the bases are base paired. "Substantially 
unpaired" means that greater than 65% of the bases of the two sequences in 
questions are not base paired, and preferably greater than 75% of the 
bases are not paired. 
F.sub.3 is preferably at least 3 bases in length, most preferably from 6 to 
12 bases in length. F.sub.4 is preferably from 3 to 5 bases in length, 
most preferably 4 bases in length. 
L.sub.1 is a short sequence of bases which preferably has the sequence 
5'-AGAA-3' when CS has the sequence 5'-NGUC-3'. Further, when L.sub.1 is 
5'-AGAA-3' and CS is 5'-NGUC-3', then the first base pair between F.sub.1 
and F.sub.4 adjacent to CS and L.sub.1 is preferably G:C or C:G (FIGS. 2 
and 6). Accordingly, in the present invention a preferred target sequence 
in a selected substrate contains the sequence 5'-BNGUC-3', wherein B is G, 
C, or U..sup.2 
The "hairpin" portion is a portion of the catalyst which folds into a 
hairpin-like configuration when the substrate-catalyst complex is modeled 
in two dimensions for minimum energy folding. This is shown in FIGS. 2 and 
6. The "hairpin" portion is not an absolute hairpin in the sense that not 
all bases of the "hairpin" portion are base-paired. Indeed, it is 
necessary for the "hairpin" portion to have at least one substantially 
unpaired region so that the catalyst can assume a tertiary structure that 
allows for better, or optimal, catalytic activity. 
The "hairpin" portion of the catalyst preferably has the sequence: 
##STR1## 
wherein, 
P.sub.1 and P.sub.4 are base sequences selected so that P.sub.1 and P.sub.4 
are substantially base paired (Helix 3, FIGS. 2 and 6). 
P.sub.1 is covalently attached to F.sub.4 ; 
S.sub.1 and S.sub.2 are sequences selected so that S.sub.1 (Loop 2) and 
S.sub.2 (Loop 4) are substantially unpaired; 
P.sub.2 and P.sub.3 are base sequences selected so that P.sub.2 and P.sub.3 
are substantially base paired (Helix 4, FIGS. 2 and 6); and 
L.sub.2 is a sequence of unpaired bases (Loop 3 ). 
"Substantially base paired" and "substantially unpaired" have the same 
meanings as discussed above. 
P.sub.1 and P.sub.4 are each preferably from 3 to 6 bases in length, and 
most preferably P.sub.1 has the sequence 5'-ACCAG-3' and P.sub.4 has the 
sequence 5'-CUGGUA-3'. It has been found that the A at the 5' end of 
5'-ACCAG-3' (underlined) is not base paired to the U at the 3' end of 
5'-CUGGMA-3' (underlined), and the unpaired A may act as a "hinge" (FIGS. 
2 and 6). 
S.sub.1 and S.sub.2 are each preferably from 4 to 9 bases in length, and 
most preferably S.sub.1 has the sequence 5'-AGAAACA-3' and S.sub.2 has the 
sequence 5'-GUAUAUUAC-3'. 
Unexpectedly, it was found that the hairpin ribozyme as constructed for an 
HIV target sequence.sup.16,28 was not as efficient as a hairpin ribozyme 
constructed with a "tetraloop" modification. 
In the prior art the preferred sequence P.sub.2 has the sequence 5'-CAC-3', 
P.sub.3 has the sequence 5'-GUG-3' and L.sub.2 has the sequence 
5'-GUU-3'..sup.16 
In the preferred embodiment of the present invention, L.sub.2, P.sub.2, 
P.sub.3 (FIGS. 2 and 6, Loop 3, Helix 4) are constructed to include the 
stable RNA hairpin sequence. 
5'-GGAC UUCG GUCC -3' (SEQ ID No:5) 
resulting in the "tetraloop" modification. As a result Helix 4 is extended 
by four base pairs over the prior art sequence listed hereinabove. 
Further, the GUU sequence of Loop 3 is replaced with the sequence UUCG. 
The resulting ribozyme is more active and more thermally stable than the 
non-modified ribozyme. 
The structure of the present invention as shown in FIG. 2 for RHVP434 and 
described hereinabove can be diagrammatically represented by the formula: 
##STR2## 
The complete sequence of the ribozyme of the preferred embodiment of the 
present invention is 
5'-UUCUUCAGAGAACAGUACCAGAGAAACACACGGACUUCG UCCGUGGUAUAUUACCUGGUA-3' (SEQ ID 
No:6). 
The structure of the present invention as shown in FIG. 6 for RHVP419 and 
described hereinabove can be diagrammatically represented by the formula: 
##STR3## 
The complete sequence of the ribozyme of the preferred embodiment of the 
present invention is 
5'-GGCUUUUAGAAGUUAACCAGAGAAACACACGGACUUCG UCCGUGGUAUAUUACCUGGUA-3' (SEQ ID 
No:11). 
The ribozyme of the present invention which cleaves the RNA of HPV can be 
used as a therapeutic agent in the treatment of HPV infections which are 
associated with genital warts and genital neoplasms. 
In the preferred embodiment, there are two methods for administering the 
therapeutic agent: gene therapy and a modification of antisense 
methodology. The therapeutic agent utilized in the present invention is 
administered in combination with other drugs or singly, consistent with 
good medical practice. The composition is administered and dosed in 
accordance with good medical practice taking into account the clinical 
condition of the individual patient, the site and method of 
administration, scheduling of administration, and other factors known to 
medical practitioners. The "effective amount" for purposes herein is thus 
determined by such considerations as are known in the art. 
(1) Human gene therapy..sup.14 The coding sequence for the HPV16 specific 
ribozyme is cloned into a vector as described herein. A U6 promoter has 
been cloned into the vector and is positioned immediately before the 
ribozyme coding region. The U6 promoter is a eukaryotic pol III promoter 
capable of driving transcription of the ribozyme using the host cell's RNA 
polymerase III..sup.6 The use of a retroviral vector for carrying the 
encoded ribozyme will aid in the integration of the ribozyme coding 
sequence within the cell's genomic DNA, thus providing long-term 
production of the anti-HPV16 ribozyme within the cell..sup.4 
To deliver the ribozyme-encoding vector to the target cells, a 
Lipofectin-based liposomal delivery system will be used. The use of 
liposomes will aid in getting the vector-ribozyme DNA to the cell without 
being degraded since the liposome acts as a protective barrier from 
nucleases..sup.25 The cells will take in the vector-containing liposomes 
via the naturally occurring process of endocytosis. The advantage of using 
the Lipofectin reagent is that it allows the liposome, once taken into the 
cell, to bypass degradation by lysosomal enzymes which is the usual fate 
of endocytic material..sup.9 In a preferred embodiment, ribozymes directed 
against either or both of E6 and E7 would be administered in combination 
with immunological agents such as LAK cells or chemotherapeutic agents 
such as cisplatin, which now has limited use in cervical cancer. Delivery 
of a ribozyme to the cervical area would be by either painting or 
injection. 
The above discussion provides a factual basis for the use of ribozymes as a 
therapy for HPV infections. The methods used with and the utility of the 
present invention can be shown by the following examples. 
EXAMPLES 
Materials and Methods: 
Enzymes and Chemicals. All restriction enzymes used were from either 
Bethesda Research Laboratories (BRL) or Boehringer Mannheim Biochemicals. 
The buffers for restriction enzymes were supplied by the manufacturer. 
T4DNA ligase and the sequencing kit were obtained from Pharmacia. The in 
vitro transcription kit and relevant enzymes were obtained from Promega. 
Bovine calf serum, antibiotics (penicillin and streptomycin), L-glutamine, 
sodium pyruvate, phosphate-buffered saline (PBS) and Dulbecco modified 
Eagle medium (DMEM) were purchased from GIBCO. 
Recombinant DNA techniques unless stated otherwise were performed as 
described in Sambrook et al..sup.18 incorporated herein by reference. 
Enzymes and Chemicals: T4 DNA Ligase and all restriction enzymes used were 
from Bethesda Research Laboratories (BRL). T7 RNA Polymerase used was 
manufactured by US Biochemicals (USB). With the exception of T7 RNA 
Polymerase, the buffers for enzymes used were supplied by the 
manufacturer. The T7 RNA Polymerase transcription buffer consisted of the 
following: 40 mM Tris pH 8.0, 6 mM MgCl.sub.2, 5 mM DTT, 1 mM Spermidine, 
1% Triton-X 100. Synthetic DNA templates used for in vitro transcriptions 
and cloning were produced using an Applied Biosystems 392 DNA synthesizer. 
Cleavage of HPV substrates was carried out in 12 mM MgCl.sub.2, 2 mM 
spermidine and 40 mM Tris pH7.5 using methods previously published..sup.11 
All reactions were carried out at 37.degree. C., with 25 nM ribozyme and 
50 nM substrate for 60 minutes unless otherwise indicated. The reference 
reaction was native (-)sTRSV sequence S17/R53 at 10 nM and 100 nM for the 
times shown..sup.11 
P.sup.32 labelling: Substrate and ribozymes were labelled with a P.sup.32 
-CTP by transcription from synthetic DNA templates using T7 RNA polymerase 
as previously described.sup.11 and reaction products separated on 15-18% 
polyacrylamide gels in 7M urea. 
Construction of the ribozyme: The ribozyme was constructed by T7 
transcription from complementary synthetic DNA templates. This was carried 
out as previously described..sup.11 
Construction of plasmids and Vectors containing RHPV: Coding and non-coding 
strands for RHPV were synthesized and HPLC purified. The strands included 
an Eco RI site, the ribozyme coding region, a poly-T termination signal 
for RNA Polymerase III, and a Bam HI site. The two strands were then 
annealed by adding an equimolar amount of each and incubating in H.sub.2 O 
at 90.degree. C. for 5 minutes, then allowed to slowly cool down to room 
temperature over a 30-minute period. The resulting double-stranded 
fragment was digested with Eco RI and Bam HI. The digestion products were 
run on an agarose gel, and the ribozyme coding fragment was isolated and 
purified. 
The plasmid PHC.sup.1 was digested with Eco RI and Bam HI, and the fragment 
was isolated and purified as above. The RHPV434 or RHVP419 fragment was 
then ligated into pHC, and the ligation mixture was used to transform 
competent DH5.alpha. bacterial cells. Single colonies were selected and 
grown in CircleGrow bacterial media, and plasmids extracted and purified 
by Sambrook's miniprep protocol..sup.18 The plasmids were screened for 
incorporation of the RHPV434 or RHVP419 insert. A colony that incorporated 
the insert was then sequenced using the Sequenase Version 2.0 enzymes and 
protocol to verify proper DNA sequence. The resulting plasmid was termed 
pHC-434 or pHC-419 respectively 
The ribozymes are cloned into a Moloney based retroviral expression vector 
for in vivo testing in human cells transformed with HVP-16. The cloning 
scheme is as follows. The ribozyme oligos are synthesized with a Pol III 
termination signal and EcoR1/BamH1 termini. These are then cloned into 
pHC,.sup.1 the standard bacterial expression vector used in a preferred 
embodiment. The ribozyme is cut out with EcoR1 HindIII and cloned into pU6 
which is a Bluescript vector containing a mouse U6 promotor..sup.6 The 
insert containing the U6 promoter is then cloned into the BamH1 site of 
pZIP-NeoSV(X)..sup.3 
pHC-434 and pMU6, a plasmid which contains an RNA polymerase III promoter 
region.sup.6 were digested with Eco RI and Hind III. The RHPV434 fragment, 
which retained the hairpin cassette region, and the pMU6 fragment were 
isolated and purified as described above. Ligation and bacterial 
transformation of the two fragments was carried out as described above. 
Colonies were screened and sequenced as described above. The resulting 
plasmid was termed pMU6-434. 
Screening of HPV Sequence (SEQ ID No:1) for cleavage site: HPV16 sequence 
data was obtained through Gen Bank. HPV16 E6 and E7 regions were inspected 
for potential target sequences as described above. All potential sites 
containing potential target sequences were tested, and ribozymes that 
showed significant catalytic activity were further developed. RHPV434 and 
RHVP419 are examples of ribozymes that showed significant catalytic 
activity. 
Example 1 
In the preferred embodiment of the present invention as shown in FIG. 2, 
Loop 3 and Helix 4 are constructed to include the stable RNA hairpin 
sequence 
5'- GGAC UUCG GUCC -3' (SEQ ID No:5) 
resulting in the "tetraloop" modification-.sup.5,27 As a result Helix 4 is 
extended by four base pairs over the non-modified sequence. Further, the 
GUU sequence of Loop 3 is replaced with the sequence UUCG. 
To determine the activity of the ribozyme, it is added to a substrate RNA 
at a ratio of 1:30 and the time course of cleavage studied as parameters 
are varied. The reaction is carried out in 12 mM MgCl.sub.2, 40 mM Tris 
pH7.5 and 2 mM spermidine over 150 minutes. For temperature dependence, 
the rate of cleavage of a ribozyme containing the tetraloop modification 
is tested over a temperature range and compared to the control reaction at 
37.degree. C. The reaction products are analyzed on polyacrylamide/urea 
gels. The bands are cut out and counted in a liquid scintillation counter. 
In the control reaction only 2% of the substrate remains after 150 min. 
indicating that the ribozyme must interact with multiple substrates during 
the course of the reaction since there were 30 times as much substrate as 
ribozyme. Further, the amount of the ribozyme remains the same and 
unaltered as expected of a catalyst. 
In the temperature dependent study of the tetraloop modification compared 
to the prior art the activity of the ribozyme was measured at 20.degree. 
C., 27.degree. C., 33.degree. C., 37.degree. C., 41.degree. C. and 
45.degree. C. 
The reaction showed a temperature dependence similar to that which would be 
expected of a reaction involving base paired RNA molecules. The Arrhenius 
plot of the data gives a temperature optimum of 37.degree. C. for the 
reaction. Higher temperatures reduce the reaction rate with a very rapid 
rate reduction about 41.degree. C. consistent with a melting out of the 
catalytic RNA structure. At 50.degree. C., no reaction was detectable. The 
reaction rate at temperatures below 37.degree. C. showed a linear 
reciprocal temperature dependence consistent with a classical lowering of 
the energy of activation for the reaction. The slope of the line in the 
Arrhenius plot gave an energy of activation of 19 Kcal/mole which is close 
to that found for catalysts fitting the "hammerhead" cleavage mechanism 
(13.1 Kcal/mole)..sup.26 
The example shows that a ribozyme with the tetraloop modification is more 
active and more thermally stable than the prior art. This form of the 
ribozyme remains active at 45.degree. C. while the nonmodified ribozyme 
lost most of its activity at this temperature. 
It was concluded from this experiment that Loop 3 does not have a conserved 
or invariant base sequence and that Helix 4 can be extended into Loop 
.degree. 3 by at least four base pairs with no loss of activity. The four 
additional base pairs in Helix 4 provide helix stabilization of this 
region. The secondary folding energy of Helix 4 and Loop 3 in the prior 
art structure is +0.6 Kcal/mole, while that of the ribozyme having the 
extended Helix 4 and Loop 3 of sequence UUCG (tetraloop) of the present 
invention was determined to be -11.1 Kcal/mole. Thus the presence of the 
tetraloop sequence increases the folding energy by 11.7 Kcal/mole. 
Example 2 
The Cleavage Reaction and optimization of helix 1 length for RHVP434. A 
cleavage study was undertaken to optimize the length of Helix 1. FIG. 3 
shows bands on a denaturing polyacrylamide gel identifying the ribozyme, 
substrate and cleavage products. Three substrates were cleaved by the 
ribozyme, each with a different length helix 1. The substrates were as 
follows: 
______________________________________ 
Helix 1 % 
Substrate Length Cleaved 
______________________________________ 
430-ACUG U*GUC CUGAAGA-444 
7 5.4 
(SEQ ID No:2) 
430-ACUG U*GUC CUGAAGAA-445 
8 6.7 
(SEQ ID No:3) 
430-ACUG U*GUC CUGAAGAAA-446 
9 6.5 
(SEQ ID No:4) 
______________________________________ 
The most efficiently cleaved substrate was that which had an 8 bp helix 1 
(SEQ ID No:3) and was used for all further studies. It is referred to as 
SHPV and the corresponding ribozyme is referred to as RHPV-434 (FIG. 2). 
Time course of cleavage. The time course for cleavage of SHPV by RHPV-434 
was done over a 180 min period (FIG. 4). The ribozyme efficiently cleaved 
the substrate to 88% completion. 
Kinetic Parameters of Cleavage. A Michaelis kinetic analysis of the 
reaction was carried out using limiting ribozyme and excess substrate for 
constant ribozyme concentration and varying substrate concentrations to 
measure initial velocities (FIG. 5). The K.sub.m for the reaction was 21 
nM and k.sub.cat or turnover number was 0.083 min.sup.-1. This gives an 
overall catalytic efficiency (kcat/Km) of 4 .mu.M.sup.-1 min.sup.-1 which 
is about 7% that of the original native hairpin sequence..sup.11 
Example 3 
The Cleavage Reaction and optimization of helix 1 length for RHVP419. A 
cleavage study was undertaken to optimize the length of Helix 1. FIG. 7 
shows bands on a denaturing polyacrylamide gel identifying the ribozyme, 
substrate and cleavage products. Four substrates were cleaved by the 
ribozyme, each with a different length helix 1. The substrates were as 
follows: 
______________________________________ 
Helix 1 % 
Substrate Length Cleaved 
______________________________________ 
415-UAAC U*GUC AAAACC-428 
6 7.5 
(SEQ ID NO:7) 
415-UAAC U*GUC AAAAGCC-429 
7 62.8 
(SEQ ID No:8) 
415-UAAC U*GUC AAAAGCCA-430 
8 12.1 
(SEQ ID No:9) 
415-UAAC U*GUC AAAAGCCAC-431 
9 28.9 
(SEQ ID No:10) 
______________________________________ 
The most efficiently cleaved substrate was that which had a 7 bp helix 1 
(SEQ ID No:8) and was used for all further studies. It is referred to as 
SHPV and, the corresponding ribozyme is referred to as RHPV-419 (FIG. 6). 
Time course of cleavage. The time course for cleavage of SHPV by RHPV-419 
was done over a 180 min period (FIG. 8). The ribozyme efficiently cleaved 
the substrate to 88% completion. 
Kinetic Parameters of Cleavage. A Michaelis-Menton kinetic analysis of the 
reaction was carried out using limiting ribozyme and excess substrate for 
constant ribozyme concentration and varying substrate concentrations to 
measure initial velocities (FIG. 9). The K.sub.m for the reaction was 98 
nm and k.sub.cat or turnover number was 0.18 min.sup.-1. This gives an 
overall catalytic efficiency (kcat/Km) of 1.8 .mu.M.sup.-1 min.sup.-1 
which is about 3% that of the original native hairpin sequence..sup.11 
Throughout this application various publications are referenced by citation 
or number. Citations for the publications referenced by number are listed 
below. The disclosures of these publications in their entireties are 
hereby incorporated by reference into this application in order to more 
fully describe the state of the art to which this invention pertains. 
The invention has been described in an illustrative manner, and it is to be 
understood that the terminology which has been used is intended to be in 
the nature of words of description rather than of limitation. 
Obviously, many modifications and variations of the present invention are 
possible in light of the above teachings. It is, therefore, to be 
understood that within the scope of the appended claims, the invention may 
be practiced otherwise than as specifically described. 
REFERENCES 
1. Altschuler, 1992, Gene, 122:85-90 
2. Anderson et al., 1994, Nuc. Acids Res. (in press) 
3. Cepko et al., 1984, Cell 37:1053-1062 
4. Chatterjee and Wong, 1993, Methods: Companion to Methods in Enzymology 
5(1):51-59 
5. Cheong et al., 1990, Nature, 346:680-82 
6. Das, 1988, EMBO J. 7(2):503-512 
7. Dipaolo et al., 1993, Critical Reviews in Oncogenesis (in press) 
8. Doeberitz et al. 
9. Felgner et al., 1993, Methods: Companion to Methods in Enzymology 
5(1):67-75 
10. Forster and Symons, 1987, Cell 49:211-220 
11. Hampel and Tritz, 1989, Biochem. 28:4929-4933 
12. Hampel et al., 1990, Nucleic Acids Research 18:299-304 
13. Haseloff and Gerlach, 1988, Nature 334:585 
14. Mulligan, 1993, Science 260:926-932 
15. Nasseri, 1991, Virol., 184:136 
16. Ojwang et al. 
17. Rossi, 1993, Methods: companion to Methods in Enzymology 5:1-5 
18. Sambrook, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor, Sections 1.25-1.28, 1.60-1.61, 1.68-1.69, 1.82-1.84, 6.9-6.13, 
6.46-6.48 
19. Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325 
20. Sedanan et al., 1991, J. Virololgy65:4860-4866 
21. Smotkin and Wettstein, 1989, J. Virology 63:1441-1447 
22. Smotkin and Wettstein, 1986, J. Virology 63:1441-1447 
23. Steele et al. 
24. Storey et al. 
25. Sullivan, 1993, Methods: Companion to Methods in Enzymology, 5(1):61-66 
26. Uhlenbeck, 1987, Nature 328:596-600 
27. Varani et al. Biochem., 30:3280-89 (1991) 
28. Yu et al 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7904 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Human papillomavirus 
(B) STRAIN: HPV16 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ACTACAATAATTCATGTATAAAACTAAGGGCGTAACCGAAATCGGTTGAACCGAAACCGG60 
TTAGTATAAAAGCAGACATTTTATGCACCAAAAGAGAACTGCAATGTTTCAGGACCCACA120 
GGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTGCAAACAACTATACATGATAT180 
AATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGC240 
TTTTCGGGATTTATGCATAGTATATAGAGATGGGAATCCATATGCTGTATGTGATAAATG300 
TTTAAAGTTTTATTCTAAAATTAGTGAGTATAGACATTATTGTTATAGTTTGTATGGAAC360 
AACATTAGAACAGCAATACAACAAACCGTTGTGTGATTTGTTAATTAGGTGTATTAACTG420 
TCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCA480 
TAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACG540 
TAGAGAAACCCAGCTGTAATCATGCATGGAGATACACCTACATTGCATGAATATATGTTA600 
GATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAG660 
GAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAAT720 
ATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACAC780 
GTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATC840 
TGTTCTCAGAAACCATAATCTACCATGGCTGATCCTGCAGGTACCAATGGGGAAGAGGGT900 
ACGGGATGTAATGGATGGTTTTATGTAGAGGCTGTAGTGGAAAAAAAAACAGGGGATGCT960 
ATATCAGATGACGAGAACGAAAATGACAGTGATACAGGTGAAGATTTGGTAGATTTTATA1020 
GTAAATGATAATGATTATTTAACACAGGCAGAAACAGAGACAGCACATGCGTTGTTTACT1080 
GCACAGGAAGCAAAACAACATAGAGATGCAGTACAGGTTCTAAAACGAAAGTATTTGGTA1140 
GTCCACTTAGTGATATTAGTGGATGTGTAGACAATAATATTAGTCCTAGATTAAAAGCTA1200 
TATGTATAGAAAAACAAAGTAGAGCTGCAAAAAGGAGATTATTTGAAAGCGAAGACAGCG1260 
GGTATGGCAATACTGAAGTGGAAACTCAGCAGATGTTACAGGTAGAAGGGCGCCATGAGA1320 
CTGAAACACCATGTAGTCAGTATAGTGGTGGAAGTGGGGGTGGTTGCAGTCAGTACAGTA1380 
GTGGAAGTGGGGGAGAGGGTGTTAGTGAAAGACACACTATATGCCAAACACCACTTACAA1440 
ATATTTTAAATGTACTAAAAACTAGTAATGCAAAGGCAGCAATGTTAGCAAAATTTAAAG1500 
AGTTATACGGGGTGAGTTTTTCAGAATTAGTAAGACCATTTAAAAGTAATAAATCAACGT1560 
GTTGCGATTGGTGTATTGCTGCATTTGGACTTACACCCAGTATAGCTGACAGTATAAAAA1620 
CACTATTACAACAATATTGTTTATATTTACACATTCAAAGTTTAGCATGTTCATGGGGAA1680 
TGGTTGTGTTACTATTAGTAAGATATAAATGTGGAAAAAATAGAGAAACAATTGAAAAAT1740 
TGCTGTCTAAACTATTATGTGTGTCTCCAATGTGTATGATGATAGAGCCTCCAAAATTGC1800 
GTAGTACAGCAGCAGCATTATATTGGTATAAAACAGGTATATCAAATATTAGTGAAGTGT1860 
ATGGAGACACGCCAGAATGGATACAAAGACAAACAGTATTACAACATAGTTTTAATGATT1920 
GTACATTTGAATTATCACAGATGGTACAATGGGCCTACGATAATGACATAGTAGACGATA1980 
GTGAAATTGCATATAAATATGCACAATTGGCAGACACTAATAGTAATGCAAGTGCCTTTC2040 
TAAAAAGTAATTCACAGGCAAAAATTGTAAAGGATTGTGCAACAATGTGTAGACATTATA2100 
AACGAGCAGAAAAAAAACAAATGAGTATGAGTCAATGGATAAAATATAGATGTGATAGGG2160 
TAGATGATGGAGGTGATTGGAAGCAAATTGTTATGTTTTTAAGGTATCAAGGTGTAGAGT2220 
TTATGTCATTTTTAACTGCATTAAAAAGATTTTTGCAAGGCATACCTAAAAAAAATTGCA2280 
TATTACTATATGGTGCAGCTAACACAGGTAAATCATTATTTGGTATGAGTTTAATGAAAT2340 
TTCTGCAAGGGTCTGTAATATGTTTTGTAAATTCTAAAAGCCATTTTTGGTTACAACCAT2400 
TAGCAGATGCCAAAATAGGTATGTTAGATGATGCTACAGTGCCCTGTTGGAACTACATAG2460 
ATGACAATTTAAGAAATGCATTGGATGGAAATTTAGTTTCTATGGATGTAAAGCATAGAC2520 
CATTGGTACAACTAAAATGCCCTCCATTATTAATTACATCTAACATTAATGCTGGTACAG2580 
ATTCTAGGTGGCCTTATTTACATAATAGATTGGTGGTGTTTACATTTCCTAATGAGTTTC2640 
CATTTGACGAAAACGGAAATCCAGTGTATGAGCTTAATGATAAGAACTGGAAATCCTTTT2700 
TCTCAAGGACGTGGTCCAGATTAAGTTTGCACGAGGACGAGGACAAGGAAAACGATGGAG2760 
ACTCTTTGCCAACGTTTAAATGTGTGTCAGGACAAAATACTAACACATTATGAAAATGAT2820 
AGTACAGACCTACGTGACCATATAGACTATTGGAAACACATGCGCCTAGAATGTGCTATT2880 
TATTACAAGGCCAGAGAAATGGGATTTAAACATATTAACCACCAAGTGGTGCCAACACTG2940 
GCTGTATCAAAGAATAAAGCATTACAAGCAATTGAACTGCAACTAACGTTAGAAACAATA3000 
TATAACTCACAATATAGTAATGAAAAGTGGACATTACAAGACGTTAGCCTTGAAGTGTAT3060 
TTAACTGCACCAACAGGATGTATAAAAAAACATGGATATACAGTGGAAGTGCAGTTTGAT3120 
GGAGACATATGCAATACAATGCATTATACAAACTGGACACATATATATATTTGTGAAGAA3180 
GCATCAGTAACTGTGGTAGAGGGTCAAGTTGACTATTATGGTTTATATTATGTTCATGAA3240 
GGAATACGAACATATTTTGTGCAGTTTAAAGATGATGCAGAAAAATATAGTAAAAATAAA3300 
GTATGGGAAGTTCATGCGGGTGGTCAGGTAATATTATGTCCTACATCTGTGTTTAGCAGC3360 
AACGAAGTATCCTCTCCTGAAATTATTAGGCAGCACTTGGCCAACCACCCCGCCGCGACC3420 
CATACCAAAGCCGTCGCCTTGGGCACCGAAGAAACACAGACGACTATCCAGCGACCAAGA3480 
TCAGAGCCAGACACCGGAAACCCCTGCCACACCACTAAGTTGTTGCACAGAGACTCAGTG3540 
GACAGTGCTCCAATCCTCACTGCATTTAACAGCTCACACAAAGGACGGATTAACTGTAAT3600 
AGTAACACTACACCCATAGTACATTTAAAAGGTGATGCTAATACTTTAAAATGTTTAAGA3660 
TATAGATTTAAAAAGCATTGTACATTGTATACTGCAGTGTCGTCTACATGGCATTGGACA3720 
GGACATAATGTAAAACATAAAAGTGCAATTGTTACACTTACATATGATAGTGAATGGCAA3780 
CGTGACCAATTTTTGTCTCAAGTTAAAATACCAAAAACTATTACAGTGTCTACTGGATTT3840 
ATGTCTATATGACAAATCTTGATACTGCATCCACAACATTACTGGCGTGCTTTTTGCTTT3900 
GCTTTGTGTGCTTTTGTGTGTCTGCCTATTAATACGTCCGCTGCTTTTGTCTGTGTCTAC3960 
ATACACATCATTAATAATATTGGTATTACTATTGTGGATAACAGCAGCCTCTGCGTTTAG4020 
GTGTTTTATTGTATATATTATATTTGTTTATATACCATTATTTTTAATACATACACATGC4080 
ACGCTTTTTAATTACATAATGTATATGTACATAATGTAATTGTTACATATAATTGTTGTA4140 
TACCATAACTTACTATTTTTTCTTTTTTATTTTCATATATAATTTTTTTTTTTGTTTGTT4200 
TGTTTGTTTTTTAATAAACTGTTATTACTTAACAATGCGACACAAACGTTCTGCAAAACG4260 
CACAAAACGTGCATCGGCTACCCAACTTTATAAAACATGCAAACAGGCAGGTACATGTCC4320 
ACCTGACATTATACCTAAGGTTGAAGGCAAAACTATTGCTGAACAAATATTACAATATGG4380 
AAGTATGGGTGTATTTTTTGGTGGGTTAGGAATTGGAACAGGGTCGGGTACAGGCGGACG4440 
CACTGGGTATATTCCATTGGGAACAAGGCCTCCCACAGCTACAGATACACTTGCTCCTGT4500 
AAGACCCCCTTTAACAGTAGATCCTGTGGGCCCTTCTGATCCTTCTATAGTTTCTTTAGT4560 
GGAAGAAACTAGTTTTATTGATGCTGGTGCACCAACATCTGTACCTTCCATTCCCCCAGA4620 
TGTATCAGGATTTAGTATTACTACTTCAACTGATACCACACCTGCTATATTAGATATTAA4680 
TAATACTGTTACTACTGTTACTACACATAATAATCCCACTTTCACTGACCCATCTGTATT4740 
GCAGCCTCCAACACCTGCAGAAACTGGAGGGCATTTTACACTTTCATCATCCACTATTAG4800 
TACACATAATTATGAAGAAATTCCTATGGATACATTTATTGTTAGCACAAACCCTAACAC4860 
AGTAACTAGTAGCACACCCATACCAGGGTCTCGCCCAGTGGCACGCCTAGGATTATATAG4920 
TCGCACAACACAACAGGTTAAAGTTGTAGACCCTGCTTTTGTAACCACTCCCACTAAACT4980 
TATTACATATGATAATCCTGCATATGAAGGTATAGATGTGGATAATACATTATATTTTTC5040 
TAGTAATGATAATAGTATTAATATAGCTCCAGATCCTGACTTTTTGGATATAGTTGCTTT5100 
ACATAGGCCAGCATTAACCTCTAGGCGTACTGGCATTAGGTACAGTAGAATTGGTAATAA5160 
ACAAACACTACGTACTCGTAGTGGAAAATCTATAGGTGCTAAGGTACATTATTATTATGA5220 
TTTAAGTACTATTGATCCTGCAGAAGAAATAGAATTACAAACTATAACACCTTCTACATA5280 
TACTACCACTTCACATGCAGCCTCACCTACTTCTATTAATAATGGATTATATGATATTTA5340 
TGCAGATGACTTTATTACAGATACTTCTACAACCCCGGTACCATCTGTACCCTCTACATC5400 
TTTATCAGGTTATATTCCTGCAAATACAACAATTCCTTTTGGTGGTGCATACAATATTCC5460 
TTTAGTATCAGGTCCTGATATACCCATTAATATAACTGACCAAGCTCCTTCATTAATTCC5520 
TATAGTTCCAGGGTCTCCACAATATACAATTATTGCTGATGCAGGTGACTTTTATTTACA5580 
TCCTAGTTATTACATGTTACGAAAACGACGTAAACGTTTACCATATTTTTTTTCAGATGT5640 
CTCTTTGGCTGCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCTAAGGTTG5700 
TAAGCACGGATGAATATGTTGCACGCACAAACATATATTATCATGCAGGAACATCCAGAC5760 
TACTTGCAGTTGGACATCCCTATTTTCCTATTAAAAAACCTAACAATAACAAAATATTAG5820 
TTCCTAAAGTATCAGGATTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATA5880 
AGTTTGGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTTTGGGCCT5940 
GTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTGGGCATTAGTGGCCATCCTT6000 
TATTAAATAAATTGGATGACACAGAAAATGCTAGTGCTTATGCAGCAAATGCAGGTGTGG6060 
ATAATAGAGAATGTATATCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCA6120 
AACCACCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCAGTAAATC6180 
CAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATTCAGGATGGTGATATGGTTC6240 
ATACTGGCTTTGGTGCTATGGACTTTACTACATTACAGGCTAACAAAAGTGAAGTTCCAC6300 
TGGATATTTGTACATCTATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCAT6360 
ATGGCGACAGCTTATTTTTTTATTTACGAAGGGAACAAATGTTTGTTAGACATTTATTTA6420 
ATAGGGCTGGTACTGTTGGTGAAAATGTACCAGACGATTTATACATTAAAGGCTCTGGGT6480 
CTACTGCAAATTTAGCCAGTTCAAATTATTTTCCTACACCTAGTGGTTCTATGGTTACCT6540 
CTGATGCCCAAATATTCAATAAACCTTATTGGTTACAACGAGCACAGGGCCACAATAATG6600 
GCATTTGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAATA6660 
TGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATATAAAAATACTAACTTTAAGG6720 
AGTACCTACGACATGGGGAGGAATATGATTTACAGTTTATTTTTCAACTGTGCAAAATAA6780 
CCTTAACTGCAGACGTTATGACATACATACATTCTATGAATTCCACTATTTTGGAGGACT6840 
GGAATTTTGGTCTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGGTTTGTAA6900 
CCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCACCTAAAGAAGATGATCCCCTTA6960 
AAAAATACACTTTTTGGGAAGTAAATTTAAAGGAAAAGTTTTCTGCAGACCTAGATCAGT7020 
TTCCTTTAGGACGCAAATTTTTACTACAAGCAGGATTGAAGGCCAAACCAAAATTTACAT7080 
TAGGAAAACGAAAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAACGCAAAA7140 
AACGTAAGCTGTAAGTATTGTATGTATGTTGAATTAGTGTTGTTTGTTGTGTATATGTTT7200 
GTATGTGCTTGTATGTGCTTGTAAATATTAAGTTGTATGTGTGTTTGTATGTATGGTATA7260 
ATAAACACGTGTGTATGTGTTTTTAAATGCTTGTGTAACTATTGTGTCATGCAACATAAA7320 
TAAACTTATTGTTTCAACACCTACTAATTGTGTTGTGGTTATTCATTGTATATAAACTAT7380 
ATTTGCTACATCCTGTTTTTGTTTTATATATACTATATTTTGTAGCGCCAGGCCCATTTT7440 
GTAGCTTCAACCGAATTCGGTTGCATGCTTTTTGGCACAAAATGTGTTTTTTTAAATAGT7500 
TCTATGTCAGCAACTATGGTTTAAACTTGTACGTTTCCTGCTTGCCATGCGTGCCAAATC7560 
CCTGTTTTCCTGACCTGCACTGCTTGCCAACCATTCCATTGTTTTTTACACTGCACTATG7620 
TGCAACTACTGAATCACTATGTACATTGTGTCATATAAAATAAATCACTATGCGCCAACG7680 
CCTTACATACCGCTGTTAGGCACATATTTTTGGCTTGTTTTAACTAACCTAATTGCATAT7740 
TTGGCATAAGGTTTAAACTTCTAAGGCCAACTAAATGTCACCCTAGTTCATACATGAACT7800 
GTGTAAAGGTTAGTCATACATTGTTCATTTGTAAAACTGCACATGGGTGTGTGCAAACCG7860 
ATTTTGGGTTACACATTTACAAGCAACTTATATAATAATACTAA7904 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ACUGUGUCCUGAAGA15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ACUGUGUCCUGAAGAA16 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ACUGUGUCCUGAAGAAA17 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GGACUUCGGUCC12 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 60 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
UUCUUCAGAGAACAGUACCAGAGAAACACACGGACUUCGUCCGUGGUAUAUUACCUGGUA60 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
UAACUGUCAAAAGC14 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
UAACUGUCAAAAGCC15 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
UAACUGUCAAAAGCCA16 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
UAACUGUCAAAAGCCAC17 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 59 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GGCUUUUAGAAGUUAACCAGAGAAACACACGGACUUCGUCCGUGGUAUAUUACCUGGUA59 
__________________________________________________________________________