CA2135643A1 - Virus resistant plants - Google Patents

Abstract

The invention features a responsive RNA molecule which encodes, in one or more protein-coding regions, a polypeptide, and which includes a regulatory domain, a substrate region, and a ribosome recognition sequence. This responsive RNA molecule has an inhibitor region in the regulatory domain, which regulatory domain is complementary to both a substrate region of the responsive RNA
molecule and to an anti-inhibitor region of a signal nucleic acid such that, in the absence of the signal nucleic acid, the inhibitor and substrate regions form a base-paired domain the formation of which reduces the level of translation of one of the protein-coding regions in the responsive RNA molecule compared to the level of translation of that one protein-coding region observed in the presence of the signal nucleic acid. The anti-inhibitor region of the signal nucleic acid is complementary in sequence to the inhibitor region of the responsive RNA molecule such that when the anti-inhibitor region is base-paired with the inhibitor region, translation of one protein-coding region of the responsive RNA is increased compared to the level of translation of that protein-coding region observed in the absence of the signal nucleic acid. The signal nucleic acid comprises part of the nucleic acid of a plant cell infecting organism.

Description

W 0 93/23532 2 1 3 ~ PCT/US93/~4240 DESCRIPTION

VIRUS RESISTANT PLANTS CONTAINING INDUCIBLE CYTOTOXIC mRNAs ~ackoround of the Invention The invention relatQ~ to methods and composition~
~uitable for producing virus reFistant plant~.
One of the most important def~n~e ~echani~ms in plant is the hypersensitive reaction. Thi~ occurs during an incompatible host pathogen (fungi, bacteria, viru~, or nematode) int OE action in which cellular changes take place that lead to~cell death. Pathogenic organisms confined to ~uch necrotic tissue~ quickly die or are restricted in t~eir ability to replicate and spread an infection.
H3-persensitive re~ponses leading ~to necrotic lesions include a 106s of per~eability of cellular membranes, i -reased re~piration, the~ accumulation and oxidation of phenolic~ compounds, and production of phytoalexins.
~Incr~asQd ~levels of ~p~cifio phenolic compounds and in uced~phytoalexins are toxic to fungi and many bacterial ~ and~ ne~at~ - pathogen . In virue di6Qases, the -~ hypersensitive respon~e~results in ~o-called local lesions - ~ in which virus- may survive-in. low concentrations for a .
considerable~ time, although the virus is confined to the lesion.
. = , Summarv of the Invention This invention features a ribozyme which acts to specifically kill plant cell~- infected with a specific virus. This in turn all:ows production of an artificial - hyp OE sensitive response in plants. This type of response can ~e- readily constructedF and appli~ to tha inhibition of ~any viral infections. For example, an artificial hypersensitive response as a result of ~iral infection in a ~obacco plant by ~obacco ~osaic virus ( ~ ) can be ~; specifically targeted. The ribozyme is constructed in such a way that a signal sequence in the viral genome 213564~
... ..
wo~3~23s32 ~ PCT/US93~4~0 stimulates the intracellular production of a toxin, e.a., an ~ ~Qli polypeptide toxic to plant cells. This creates a hypersensitive response in the plants by killing cells infected with a virus. The secondary structure of the 3' end of the TMV positive strand RNA genome has been very well characterized, and thus the determination of non-base paired regions as possible signal sequences is readily performed.
Specifically, the invention features an RNA molecule, termèd a responsive RNA molecule which, when present in a plant cell, responds to the presence of other nucleic acids. By Nresponds" is meant that the respon6ive RNA
molecule will be translated to form one or more polypeptides in the presen¢e of certain nucleic acids ~15 (which can hybridize to the responsive RNA) and will not be significantly translated to form these polypeptides in the absence of such nucleic acids. Such a responsive RNA
molecule will gen rally encode one or more polypeptide molecules, the production of which depends on translation 20~ of that respon ive RNA molecule. Generally, translation of the responsive RNA molecule, and thus production of polypeptide, will not occur in any particular ce}l unless a pecific nucleic acid, termed a signal nucleic acid, is also pres-nt within that cell.
A responsive RNA can be used to kill or injure sp cific cells within a population of cells. For example, a responsive RNA may encod a toxin molecule which is produced from the responsive RNA only when the responsive RNA molecule within a given cell is exposed to a signal - 30 nucleic- acid indicative of a condition (~, infection with a_-harmful virus such as TMV) r~quiring that the cell `
be kiLled. More specifically, the responsive RNA molecule may encode a cytotoxic protein such as cholera toxin, diphtheria toxin, ricin and the hok, gef, RelF or flm gene products of E. ÇQli, and translation of the responsive RNA
molecule and production of cytotoxic protein occurs only when the responsive RNA molecule is present within a cell ~13~643 wos3~23s32 PCT/US93/04240 =
which is infected with TMV. Here, an RNA molecule spe-cific to TMV or a portion of the TNV RNA genome serves as the signal nucleic acid and interacts with the responsive RNA molecule to allow translation of the tox~n-encoding sequences of the responsive RNA molecule.
A responsive RNA molecule is produced by designing a polypeptide-encoding RNA which, in the absence of a signal nucleic acid, has a structure which prevents translation.
One type of respon~ive RNA molecule can fold to form a base-paired domain, e.~., which, when suffici¢ntly stable, prevents translation by preventing the translational mac-hinery of a cell from reading the nucleotide sequence of the RNA. A specific example of a responsive RNA molecule of this type has a domain which encodes the desired polypeptide (or ~protein-coding region~) and a regulatory do~in (i.e., a do ain which includes regulatory elements including an i-nhibitor-region, invert-d repeats and nucle-ation regions;. ~ The re~ulatory domain may be located anywhere in the reJponsive RNA molecule 80 long as the segyence of the elements of the regulatory domain are se1ect~d so as not to interfere with the activity of the coded polyp-ptide. The inhibitor region is complementary n sequence to both a substra-t~ region (which can include ., portions of ~either the~protein-coding region and/or a 2-5 leader region which is the non-translated RNA 5' of the ; protein-coding region or portions-of-the ~NA genome of an RNA viru~) and to a region-of the signal nucleic acid referred to as an anti-inhibitor- region. In the absence of the signal nucieic acid, the-inh-ibitorv region of the res~onsive RNA molecule hybridizes to the substrate region ~ - of a responsive RNA molecule- fQrming an intramolecular base-paired domain which pr-vent~ or reduces translation.
When the signal nucleic acid is pre~nt, the anti-inhibitor~region compet~s with the substrate region for binding to the inhibitor region. FormatiQn of an intermolecular base-paired domain between the anti-inhibitor region of the signal nucleic acid and the ~135643 ~i W093/23~32 PCT/US93/04240 inhibitor region of the responsive RNA prevents formation of a base-paired region with the protein-coding region;
under these circumstances the protein-coding region(s) can be translated.
A second type of responsive-RNA molecule has an intervening sequence or "intron", the presence of which prevents translation of one or more ~exonsn. Introns do not code for the desired polypeptides. Segments of the RNA which code for desired polypeptides are called "exons"
as are non-coding sequences (e.a., the leader region, secretory sianal sequences, poly(A) tails, and the like) that remain after the splicing reaction. This second type of responsive RNA molecule is desianed so that it can undergo a splicing reaction under desired conditions ~ 15 ~çhg~! in the presence of a specific RNA molecule) which -~ r--ov~s~the intron and joins the two flanking portions of the~RNA molecule, thus forming a ~olecu}e which is the prop r- template for the~ active polypeptide. It is the regulation of this splicing reaction which in turn regu-lates translation. This second type of responsive RNA
~olecule is similar to the first type of responsive RNA -~olecule in that it has an inhibitor region which is comple~ent~ry-- in --s~quence to both the anti-inhibitor region Or a signal nucleic acid ~nd to a substrate region within the responsive RNA molecule. In this second type of responsive--RNA,-the sub6trate region i8 not neces~arily ¦
part of an-exon, but rath r contains a region which is essential to the self-splicing reaction. When the substrate region is base-paired to the inhibitor region, ~- 30 the self-splicing reàction cannot occur, thus translation is prevented-.- In contrast, when a signal nucleic acid is ~`
present, its anti-inhibitor reg~on hybridizes to the inhibitor region of the responsive RNA forming an intermoleculnr base-paired domain, which prevents intramolecular base-pairing between the inhibitor region and the substrate region. Under these circumstances, the substrate region is free to participate in the splicing I
.... , . ... .. , , .:::

~13S643 reaction, the intron is removed, and translation of properly joined exons can oc ~ .
Thus, in a f irst aspect the invention features a responsive RNA molecule which encodes, in one or more protein-coding regions, a polypeptide, and which includes a regulatory do~ain, a s~b~trate region, and a ribosome recognition ~e~uence, e.g., a ribosome binding site, a tran~lation initiation site, a~d all non-coding regions necessary for the tran~lation of an RNA. This responsive RN~ molecule has an inhibitor region in the regulatory domain which is compl~mentary to both a substrate region of the responsive RN~ molecule a~d to an anti-inhibitor reqion of a signal nucleic acid such that, in the absence of the signal nucleic acid, the inhibitor and substrate regions form a ~ase-paired domain which reduce~ the level of translation of the responsive RNA mole~ule compared to that level observed in ~he presence o~ a signal nucleic acid. The signal RNA is chosen from one present only in plant cells which mufit be selectively kill~d, e.g., TMV
genomic RNA or m~NA.
The Hregulatory domainN is a region of the responsive RNA molecule which will regnlate the level of translation of the responsive RNA molecule dependen~ upon the presance of the signal nucleic acid. The regulatory region includes the inhibitor xegion, inverted _repeats and n~cleation regions. A "ribosome recogni~ion sequence" is a region of an RNA molecule that is-reguired in order for translation to begin at a given initiation codon (typically AUG). Such a si~_e is re~ognized by a riboso~e and bound by the ribosome prior to---th~ init~ation of translation of the RNA. In procaryotes, the ribosome recognition se~uence is a ri~os~me binding site and includes a purine-rich sequence centered about 10 nucleotides 5' to the initiation codon (Shine and Dalgarno, ~roc. Natl. ~cad. Sci. USA ~1:1342, 1974). For eucaryotes, the sequence A/G NNAUGG described by Kozak (Kozak, J. Cell Biol~ 108:229, 1989~ is the minimal 2135~3 W093/23~32 PCT/US93/04240 ribosome recognition ~equence required for initiation of translation. This sequence includes the AUG initiation codon. -:
The "signal nucleic acid" is a nucleic acid (e.a., a viral RNA~ which is indicative of a condition under which it is desirable to produce the polypeptide encoded by the responsive RNA molecule.
A "base-paired" domain is a region over which the nucleotides of two regions of nucleic acid are hydrogen-bonded to each other. The term includes bonding of lessthan all cont-iguous nucleotides of such region~.
The "substrate region" is a region of the responsive RNA molecule which w~en base-paired reduaes the level of translation of one or more of the protein coding regions 15 in th~ responsi~e RNA ~olecule.
The ~inhibitor r~gion" is a region of the r~sponsive RNA molecule which when base-paired to the substrate region reduces the level of tran~lation of one or more I -protein-coding regions in the responsive RNA molecule.
The "anti-inhibitor region" is a region of the signal nucleic acid which when ~ase-paired to the inhibitory region incre~ses the level of translation of one or more protein-codIng regions of the responsive RNA molecule compared to that observed in the absence of the signal nucleic acid molecule. These three regions interact to .
regulate the~l~vel of translation of the responsive RNA
molecule and are selected to i~s~re appropriate levels of polypeptide production dependent upon the presence of the signal nucleic ~cid.
An "indicator gene" include~ a coding region whose expression ca~- b~ easily identified. For example, the , genes enc~ding luciferase, ~-glucuronidase, or chloramphenicol aetyltransferasa. ' :-By n-appropriate level" is meant that in the absence ~:
35 of the signal nucleic acid the level of polypeptide is ~:
sufficiently low to have little or no effect on the `-physiology of the cell, and in the presence of the signal ;~

~135fi~3 w093/23532 PCT/US93/04240 nucleic acid the level of polypeptide is sufficiently high to reduce viability of the cell. The level of translation of the responsive RNA can be determined by standard procedures. Genera}ly, a low level of translation is one in which less than 0.1% of the polypeptide produced by a cell is polypeptide encoded by the responsive RNA
molecule.
In preferred embodiments, the sub~trate region is part of an exon or a leader region or overlaps the junction betw~en the two (which includes the ribosome recognition 6equence, and the initiation codon), or includes a region necessary for the self-splicing reaction. ~;~
In eucaryotic cells, the 40S subunit of the 15 eucaryotic ribosome bind~;at the 5'-end of a capped mRNA I ;;
and~ ~scans~ down the ~e~age `in search of the first ~ initiation ~codon (see generally Kozak, J. Cell. Biol.
- ~ 08:229,~1989). In thi~ process, all but extremely stable i hybrid~ (i.e., t~o~e~h~ving a free energy of formation of 20 ~-50 kcal/mol) are unwound and 6cannQd through (Xozak, -~;~
Proc. Natl. Acad. Sci. USA ~:2850, 1986). Thus, to inhibit ~canning of the 40S subunit to the translation ---~
initiation site, the-inhibitor region ~ust gen-rally form an~exten~ive bylrid with th~ ~ub~trate region (which may 2-5 include thé~ ribo~ome recognition sequence and/or the initiation codon) in-~which the base-pa-ire~:-region has a j fr-e energy ~of formati~n th~t is~ -50 kcal/mol or lower.
~hus, it is preferred that the inhibitor region be located downs~ream (3') of the r~bo~ome recognition-sequence (in 3;0 the exon or perhaps nearer the 3' end o`~ the me~sage) so that the ~nteraction between the inhibitor region and the ~nti-inhibitor ~ignal RNA (which wou~~ha~~a similar if not lower free energy of rormation) wouid not al$o prevent movement of the- 40S ribosomal subunit to the initiation ~ite ~see, Figs. ~F, lG, and lH). Accordingly, in a plant eucaryotic system, ha~ing the self-splicing intron interrupt the protein-coding region is preferred.

~1356'~3 wo93/23s32 ~ t ` PCT/US93/04~0 As used herein an "intron~ is a domain of the responsive RNA molecule which is separate from the exons.
Pr~ferably the intron is an RNA molecule having catalytic activity including RNA cleavage and ligation activity. It is preferred that such an intron be able to self splice and thus is chosen fro~ a group I or group II intron, such as that present in Tetrah~mena thermoDhilz.
In more preferred embodiments, the re~ponsive RNA
molecule is purified, and the responsive RNA encodes a lo polypeptide which modifies cell viability, cell proliferation, transcription of DNA, translation of RNA, or replication of DNA, ~3~, the responsive RNA. molecule encodes a polypeptide which has diphtheria toxin acti~ity or ribonuclease activity.
,, ~Purified RNA~ is RNA isolated from one or more co~ponents~ of the e mironment in which it naturally occurs. For ex~mple, the RNA is present in a cell in which it does not naturally oc as is the case with foreign gen-s expressed in transgenic plants. Preferably it i8-: provided~as a ho~ogeneous solution of nucleic acid.
In other preferred embodiments, the substrate region includes the 5'-splice junction of the intron; the intron reduces the leveL of translation of the exons~compared to ~ the level of translation in the absence of the intron; the intron is located~ ~etw en the ribosome recognition equence and a 5-'-mos`t exon or between two exons. Even more preferably, the intron overlaps at its 5'-end a 5'-splice junction, and at its 3'-end a 3'-splice junction;
the intron catalyzes two cleavage reactions, one within the 5'-splice -junction and one within the 3'-splice junction; the intr n is a self-spliclng intron; the substrate region:IncIudes the 5'-splice ~unction; and the inhibitor region interferes with the cleavage reaction within the 5'-splice junction.
A "5'-splice junction~ refers to the sequence overlapping or abutting the 5'-end of an intron which is required for a splicing reaction. A "3'-splice junction"

6 4 3 - `
Wos3t23s32 PCT/US93/04 refers to the sequence at the 3'-end of an intron which is required for a splicing reaction. Such splice junctions overlap the ends of a self-splicing intron such as those bordering the intervening sequence of TetrahYmena thermo~hila.
A "self-splicing intron" is a piece of RNA which contains all of the sequences required except for the necessary abutting splice junction sequences for the intron to excise itself from a larger piece of RNA and to join the two pieces of RNA that flanked the intron prior to the excision reaction. That is, the intron is able to cleave and liga~- two portions of an RNA molecule.
In yet more ~referred embodi~ents, the signal nucleic acid is single stranded, çhg,, it is viral RNA.
Examples of responsive RNA include Tetrahvmena RNA
wh~ch has been ~odified, for example, by nucleotide changes at positions -14, -19, -21, -22, -23 and/or -24 relative to the 5'-spiice site.
In a reIated aspect the invention features a method for interferin~ with the growth of a cell harboring a signal nucleic acid by introducing a responsive RNA
molecule as describ d above into the cell.
~-- ; In another r-lated aspect, the inven~ion features a DNA mol~cule~encoding the above responsive~RNA molecules.
This invention~is~based upon the non-natural use of the hypersensitive response with RNA -sel~-~plicing dQpendent on the presence of specific RNA ~quences.
Ribozymes designed to undergo self-pr~ce~sing when hybridized to unique viral sequences offer a highly sèlective switch which will bloc~ translation of a~toxic peptide until the plant is inf-cted with a-spQcific virus.
Transgenic plants encoding such RNA wil~-~n-ot- produce a ;~ foreign or leth~l protein until it is n~eded to combat a viral infection. Even when the toxic polypeptide in production is limited to the cells infected by a specific virus, and the death of infected cells will terminate production of the toxic protein. This application of RNA

!

~13~6~
W093/23532 PCT/US93/04~0 , . I, , proce~sing technology significantly enhances the natural defense mechanism of the plant, and does not produce high levels of a foreign protein. Only low levels of the self-splicing RNA coding for a toxin are required, and as a result, little of the plant's resources are needed to produce an antiviral response.
~ ransgenic plants can be produced which are resistant to viral infections, and thus will have less crop damage and provide higher yields. This invention can be applied to all plant viral sy6tems in which the viral genetic sequences are known or deciphered, and the infected plant can be transformed.
Other features and advantages of the invention will be apparent from the following description of the preferred eD~odiments thereof, and from the claims.

~cription of the Preferred Embodiments The drawings are first briefly described.

B~ef Description of the Drawinas Figs. 1, lA and lB are schematic drawings of a responsive RNA molecule. The thin line represents the leader region, the thick }ine represents a protein-coding region, a series of short vertical lines indic~tes a base-paired domain, and the boxes above and below these lines indicate various features-of the RNA. Specifically, in Fig. LA the responsive RNA is drawn so as to depict intramolecular ba~e-pairing which prevents translation;
and in Fig. lB the responsive RNA molecule is depicted as hybridized to a signal-nucleic acid.
Fig. lC depicts a_second variation of this type of responsive RNA molecule,- in Fig. lD the responsive RNA
molecule is drawn to depict the intramolecular base-pairing that prevents translation; and in Fig. lE the respon~ive RNA molecule is hybridized to a signal nucleic acid.

WOg3/2353~ ~ ~CT/US93/04240 -~ ig. lF depicts a ~ ird variation of a responsi~e RNAmolecule; in Fig. lG this responsive RNA molecule is drawn to show the intra~olecular ba6e-pairing which prevents translation; and in Fig. lH the responsive RNA ~olecule is hybridized to a signal nucleic acid.
Figs. 2, 2A, 2B~ and 2C are schematic drawing~ of a responsive ~N~ molecul~ which includes a self-splicing intron. The thin line represents a leader region, the broken line represents a self-splicing intron, the thick line represents an exon, a series of short vertical lines indicates a ba~e-paired doma~n, and the boxes abov~ and below these lines represent various ~eatures of the RNA.
Specifically, in Fig. 2A the responsive RNA molecule is drawn o as to depict the intramol~cular bas~-pairing whiçh pr~vents self-splicing; ~n Fig. 2B the responsive RN~ molecule is depicted as hybridized to a ~ignal nucleic acid; and Fig. 2C depicts the spliced molecule produced by the self-splicing reaction.
Figs. 2D, 2E, 2F and 2G depict a variation of the type of responsive RNA molecule shown in Figs. 2-2C. In Fig. 2D, a self-splicing intron ~eparates the polypeptide-coding sequence in the responsive RNA molecule; in Fig.
2E, the responsive RNA ~olecule is drawn-to depict the intramolecular base-pairing which prevents self-æplicing;
in Fig. 2F, the responsive RNA molecule i8 hybridized to a ~ignal nucleic acid; and in Fig. 2G,- the spliced ~olecule produced by the self-splicing reaction is depicted.
Fig. 3 depicts P(l) and P(-1) stem~loop structures at or just upstream of the 5' exon-intervening sequence (IVS) junction of Tetrahymena thermophila. The IVS.(uppercase) contains the internal guide se~uence (boxed) which can hybridize with the end of the S' exon (lowercase) to form the P(1) stem-loop, the conformation required at the 5'-splice site (shown by filled-in tsiangle) for self-splicinq. The alternative structure P(-1), which does not support self-splicing, is formed by hybridization between ~1~51;~
W093~23s32 PCT/US93/04~0 ~ i~
a portion of the P(1) stem (boldface) with an upstream 5' exon sequence (overlined). The sequence shown at the top is that ~or RNA from the parent plasmid pTETBLU. The lower three ~NA structures represent modified P(-1) stem-loops from three mutant plasmids that were made bysequence changes (shaded~ in the 5' exon. Calculated free energies at 37C for each of these structures are given.
Fig. 4 is a copy of a photograph of a polyacrylamide gel showing the results of n vitro transcription reactions carried out in the presence of L~32P]CTP using the parent plasmid (pTETBLU) and the three splicing mutants (pTET14, pTET1419, pTET21-24) as templates. Each set of three lanes represents the transcription products before (O) and after ~15 or 60 min) the change to splicing condition~. The template used is given above each set of lanes, and the restriction enzyme used to linearize the template is shown at the top. For this and the following two figures, FL denote~ the full-length precur~or RNA and LE indicate~ ligated ~xons. The positions of linear rvs RNA (L-IVS) and circular IVS RNA (C-IVS) are also indicated, as are the shortened forms of L-IVS in which 15 or- 19 nt have -been removed from the 5' end by the circularization reactio~ (~-15 and L-19, respectively).
An asterisk denotes an RNA thought to be the product of 3'-splice site hydrolysis_(~.e., a 5' exon-IVS fragment).
An as yet unidentiiled--small RNA product is also indicated (<) - .
Fig. 5 is a copy of a photograph of a polyacrylamide gel showing the results of experiments in which gel-purified pTET1419 RNA was incubated under splicing - conditions in the a~sence (0) or presence of ~he given con~entrations o~- either of two signal RNAs (4S or 4S3) for 15 or 60 min. The resultant products were analyzed on a 4% denaturing polyacrylamide gel and are indîcated in 35 Fig. 5.
Fig. 6 is a copy of a photograph of a polyacrylamide gel showing the results of experiments in which gel~

~13~ 13 w093/23~32 PCT/US93/04240 purified pTETBLU RNA or pTET21-24 RNA (10 nM) was incubated in splicing buffer at 4 or 37C. Where indicated, Mg2' was added to 5 mM to initiate the æplicing reaction. For pTET21-24, splicing was initiated in the absence or presence of either of two signal RNAs specific for the pTET21-24 sequence (8S4 or 12S). When present, the concentration of the signal RNA is 1 ~M. The resulting products, analyzed on a 4~ denaturing polyacrylamide gel, are labeled as in Fig. 4. Templates used for transcription were linearized with either EcoRI
or ~HI as indicated at the top. An additional product seen when the EçQRI-runoff precursor is incubated under splicing conditions in the presence of, signal RNA is indicated with a dot. A short RNA product (~) seen when pIE321-24 is incubated under splicing co~ditions in the ~absence of a signal RNA is mark d with an arrowh ad. This s~me RNA product i~ also visualized in Fig. 4.

Transaenic Plants Responsive RNA ~olecules, e.a., ribozymes can be , 20 developed which undergo a self-splicing reaction when a target s qu nce in the RNA is b~se paired to an RNA signal sequence. This~ RNA enables th- signal sequence~ nduced relQase of intron sequ~nc-s inhibiting~ the corre_t tr~n~l~tion of a toxic polypeptide. Signal ~equence inducéd ,RNA splicing can thus be used to~-s lectively express a toxic polypeptide. This type of RNA or'nkiller ribozyme" is useful in the selective death of specific plant cells. Such ribozymes can be introduced int~ plants ~ on standard vectors as DNA encoding the de~ired,,ribozyme.
The ~killer ribozyme" provides the opportunity to produce an ~rti~icial hypersensitive response in~plants.
The ~killer ribozyme~ is constructed in such a way that a signal sequence in viral, transcripts or the viral RNA
genome stimulates the intracellular production of an ~ ÇQli polypeptide toxic to plant cells. This creates a hypersensitive response in transgenic plants by killing ..

~13~3 wo93/23s32 PCT/US93/04240 cells infected with a virus. When a- hypersen~itive response is induced by viral infection, either a necrotic lesion will form, or if the response is extremely efficient the single infected cell will die before limited spread of infection to adjacent cell~.
Thus, transgenic plants can be produced which mimic a hypersensitive respon~e as a result of viral infection.
This will result in inhibition of the spread and replication of virus in plants and a mode of producing viral re~istant plants.
In order to design such a ribozyme, different signal ~equences, non-signal induced splicing of the ribozyme, and the toxicity of the appropriate polypeptide can be assay~d in, e.~., a tobacco protoplast system.
Protoplast~ can be induced to take up both expres~ion vec*ors~ containing the ribosy~e construct and viral particles. Instead of the toxic peptide sequences, the ribozyme may be constructed with an indicator gene which when expre~ed will be translated after signal seguences ~20 ~(viral~g~no~ic RNA) induce splicing of the ribozy~e. This ~will provide the c~pac~ty to easily test different signal . .-sequ~nces and deter~ine the degree to, which non-signal ` .-induced splicing occurs.
Information deri~ed~from the protoplast assayC can be ~25 u~ea in de~ loping ~killer ribozyme~ optimiz~d for signal seguence and low nonsignal-induced splicing. Utilizing this information, constructs can be developed for the --production of transgenic tobacco plants, which respond to TMV infection with an ar~ificial hypersensitive response. ~
. ..
30 Res~onsive RNA Molecules `
Respon~ive RNA--moiQcules are generally described above. Below are presented specific examples to illustrate these mQlecules to tho~e of ordinary skill in the art. These examples are not limiting to this ~
35 invention. -~13~fi~ :
W093/23s32 PCT/US93/04240 Exampl~_L~_~ç~onsive R~A mol~cules without introns A firct type of responsive RNA molecule is illustrated in Fig. 1. one portion of this molecule, the protein-coding region encodes a polypeptide whose production is desired only in the presence of a signal nucleic acid. Another portion of the molecule, the regulatory domain, includes an inhibitor region which is complementary in sequence to a substrate region within the protein-coding region. The inhibitor region can ba~e pair with the substrate region to form a base-paired domain which blocks translation of the protein-coding region.
The substrate region can be a part of the protein-coding region, part of the lQader region, or overlap the junction between the two.
Referrin~ to Fig.~ l, re~pon~ive RNA molec~le 10 has a 5'-end 12, and a 3'-end 14. Adjacent to 5'-end 12 is a leader region 26 and regulatory domain 16; adjacent to 3'-end 14 i8 a prot~in-coding region 18. Within regulatory domain 16 is an inhibitor region 20; within protein-coding region 18 is a substrate region 22. At the 5' of protein-coding region 18 is a ribosome recognition sequenc~ 21 and an initiation codon 23.
Referring to Fig. lA, inhibitor region 2~ hybridizes to s~bstrate region 22 to form a base-paired domain 28.
Such base-pairing within responsive RNA molecule lO
inhibits translation of the protein-coding reg-ion-o-f`the responsive RNA molecule. I
The inhibition of translation is relieved by the presencs o~ a signal nucleic acid, a region o~ which, referred to as the anti-inhibitor, is complementary to the inhibitor region of the responsive RNA. The anti-inhibitor region of the signal nucleic acid competes with the substrate region of the responsive RNA ~olecule for hy~ridization (base pairing) with the inhibitor region of the responsive RNA molecule. Under these circumstances there is-no base pair formation with the substrate region, ~3~6~
Wos3/23~32 PCT/US93/04 translation occurs and the desired polypeptide is produced.
For example, referring to Fig. lB, signal nucleic acid 30 has a 3'-end 32, a 5'-end 34, and an anti-S inhibitor region 36 complementary in sequence to inhibitorregion 20 of responsive RNA molecule 10. Hybridization of - anti-inhibitor region 36 with inhibitor region 20 forms base-paired domain 38 and prevent~ hybridization of inhibitor region 20 to ~ubstrate region 22. Under the~e circumstances, translation of protein-coding region 18 occurs.
In a variation of this type of re ponsive RNA
molecule, the substrate region is not entirely contained within the protein-coding region~but extends upstream of the protein-coding region into the le~der règion.
Specifically, the rQsponsive RNA molecule depicted in Fig.
lC ha~ a~ -ub tr~te~region 22 which includes the ribosome recognition s~gu nce 21 and the initiation codon 23.
Referring~ to Fig. lD, ub-trate~ region 22 base-pairs to 20~inhibi~or region 20 forming intramolecular base-paired r~gion 28~. In~a~ procaryotic system, this configuration phy&ically blocks a ribosome from interacting with the ribo-ome binding site~-and-~the initiation site, and tr~n~lation i~ inhibited. Referring to Fig. lE, the anti-25~ inhibitor region 36 of the ignal nucleic acid 30 is hybridized to the inhibitor-reqion 20 to for~ base-paired region 38. In this configuration, a procaryotic ribosome initiates translation and the desired polypeptide is produced. - -In another variation--o~ this typ~ of responsive RNA
- -molecule, the inhibitor--region is located downstream of the substrate region. Ih~_inhibitor region can be within the protein-coding region it~elf, as diagrammed in this ~igure, or located in a region 3' of the protein-coding region. In Fig. lF, the responsive RNA molecule is depicted as having a substrate region 22 that includes ribosome recognition sequence 21 and initiation codon 23 , ~135~43 ;

and has an inhibitor region 20 located 3' of the substrat~
region. Referring to Fig. lG, the inhibitor region, 20, base-pairs with the substrate region 22 forming intra- :
molecular base-paired region 28. In this configuration, S a scanning euca~yotic ribosomal subunit cannot i~vade or bind to the base-paired domain to initiate translation provided ~his ba~epairing interaction is suf f iciently strong. In Fig. 1~, the anti-inhibitor region 36 of signal nucleic acid 30 i8 hybridized to inhibitor region 20 forming base-paired region 38. A eucaryotic ribosome can scan to the proper initiation codon (provided there are no other upstream ini~iation codon.) and initiate translation. Translation of the polypeptide occurs, wi~h disruption of base-paired region 38 by the translating ri~osome.
Since the inhibitor region of the re~ponsive RNA must be complementary to both the substrate region of the responsive RNA, and the anti-inh.~itor region of the target nucl~ic acid, the ~equences of these three region~
must be chosen to allow suitable regulation of translation - of the responsive RNA. This does not mean that the sequence of the substrate region must be identical to the -~egu~nce of the anti-inhibitor region. Neither o~ the t~o base-paired domains which can form need to be perfectly base-paired ~ ~ , all contiguous bases along the domai~s are base-paired), nor do they have to be the ~ame length.~-There is flexibility in the selection of the anti-inhibitor region so long as the region is specific enough to indicate when translation must occur. For example, if the signal to which the responsive RNA responds i8 the presence of TMV genome or-transcripts within a cell, any specific nucleic acid sequence of ~NV could be chosen,~â~d of cours~, one is lLmit~d in s~lecting a nucleic acid ~equence present in TMV. The sequence of the substrate reg~on is cho~en to create a responsive RNA molecule which produces a biologically active polypeptide. Since the substrate region may include portions of a protein-coding ~1356~3 W093J~3~32 ' ; PCT/USg3/04 region, any modification of its sequence must preserve a significant amount of the activity of the encoded poly~
peptide. The dQgeneracy of the genetic code allows for changes in the sequence of the protein-coding region which do not affect the sequence of the encoded polypeptide.
Because guanosine can base-pair with uridine as well as with cytosine there is additional flexibility in the sequences which can be used. In addition, since conser-vative amino acid changes at one or more positions in proteins often do not eliminate activity of the protein the number of usefu} sequences is increased substantially.
The base-paired domain formed by hybridization of the inhibitor region to the substrate region must be stable enough so that it will not b~ disrupted by nucleic acids other than the signal nucleic acid, which may also be pre~ent within the cell. For example, if the inhibitor region and the ~ubstrate region are comple~entary over only four contiguous nucleotides, any single stranded nucleic a¢id th~t includes that four base sequence could conpete with the substrate region for hybridization to the inhibitor region, and if the nucleic acid including this sequence was present at a high enough concentration inhibition of translation wouldL be relieved. Generally, ' the~base-paired dom~in for~ed~by the hybridization of the~
sub~trate region to the inhibitor region should include at least 12~, and preferably~'l5~ -contiguouæ nucleotides in order' for the molecule -to respond to only the signal nucleic acid.
The responsive RNA molecule can include a region that will allow the signal nucleic acid to more readily hybridize to the inhibitor region-. This additional region is called a nucleation reg~on'~and consi~ts of a number of _ nucleotides immediately ad~acent to the inhibitor region and complementary to the seguence of the signal nucleic acid such that the nucleation region and the inhibitor together form a region of extended complementarity with the signal nucleic acid. The nucleation region pro~ides wo g3/23s32 ~ 1 3 ~ 6 ~ 3 PCT/~S93/04~40 a single stranded region that is readily available for hybridization to the ~ignal nucleic acid. Base-pair formation over ~ is region will tend to favor displace~ent of the substrate region from the inhibitor region by positioning the an~i-inhibitor region corr~ctly for hybridization to the inhibitor region. In addition, such a nucleation region will increase the sta~ility of the base-paired region formed with a signal nucleic acid.
The regulatory domain may also include a region that will disfavor hybridization of non~specific nucleic acids ( e., nucleic acids other than the signal nucleic acid) to the region immediately ad3 aaent to the inhibitor domain. This r~gion is referred to as an inverted repeat and can fold to form a hairpin structure.
The detailed nature of the inhi~itor region, ~he sub8trate region, and the anti-inhibitor region wil depend, in part, on the extent that translation is to be regulated. The more stable the intramolecular base-paired domain formed by hybridiza~ion of the inhibitor region to the substrate region, the more translation will be inhibited. For RNA-RNA duplexes, the stability of a base-paired domain depends on the number of nucleotides actually ba~e-pairad within a contiguous region- of .-nucleotides, the number o~ mismatches within a gener~lly ~ase-paired domain, and the nucleotide composition of the _ bas~-paired domain. I~tramole~ular ~ase-pair formation-depends on the distance between the two regions to be base-paired. For example, when there are too few nucleotides between the two regions, tor~ional-type.
constraints can prevent base pair formation. ~hose in the ar~ are well aware of how these parameters can be adjust~.
in order to make a more or less stable baæe-paired domaln.
The stability of the intr~molecular base-paired domain can be adjusted dependent upon the l~vel of translation that is desired at any given level of signal nucleic acid. The level of tran~lation depends on the proportion of responsive RNA molecules in which the inhibitor region is ~13~43 w093/23532 PCT/US93/04240 :".

hybridized to the substrate region. This proportion, in the presence of the signal nucleic acid, depends on the proportion of the re~ponsive RNA molecules in which the inhibitor region is hybridized to the anti-inhibitor region of the signal nucleic acid. Those in the art wil}
appreciate that the amount of each duplex which foras depends on the relative stability of the two duplexes as well as the amount of signal nucleic acid and responsive RNA present in a given cell. If a highly toxic molecule is encoded by the responsive RNA then a high degree of regulation is required. For examp}e ! if the active subunit of cholera toxin is encoded, only a few molecules are reguired to kill a cell. In this ca~e translation ~u~t be completely inhibited in the absence of signal nucleic acid. Thi~ is be~t insured by having almost co~pl~te complementarity of the substrate and the inhibitor region~, e.., 85% comple~entarity of a 20 nucleotide region. Expres~ion occurs only when a highly - co~pIQ~entary ~ignal RNA i6 present, having e.sL, 100%
complementarity to the inhibitor region over a 25 nucleotid- region.
The inhibitor region may be on~the 5'-~ide or the 3'-side of the protein-coding E~gion or within the protein-coding region itself. If the responsive RNA molecule is ~ubject to exonucleolytic degradation, this should be taken into w count when designing-the ~olecule. Thu~, if the ~olecule i8 degradod beginning at the 3'-end it would be be~t to locate the inhibitor region at the 5'-end of the molecule in order to prevent formation of a molecule containing all of the ~eguences reguired for translation but lacking an inhibitor reg~on~
~ , - Exam~le 2: ~çsponsive RNA molecules with self-splicina introns A second type of responsive RNA molecule includes a 3S ~elf-splicing ~ntron which prevents production of the desired polypeptide. The intron can be removed by a :, ,~ c W093t~3~32 ~ 1 3 S fi ~ 3 PCT/~JS93/04240 splicing reaction, and the spliced molecule serves as a template for the production of the desired polypeptide.
A signal nucleic acid regul~tes translation of this type of responsive RNA molecule, but the regulation is achieved indirectly by using the signal nucleic acid to regulate the splicing reaction. In order for this type of regulation to work the responsive RNA molecule mu~t, in the absence of the signal nucleic acid, fold so as to form an intramolecular base-paired domain which prevents splicing. In the presence of the signal nucleic acid an alternati~e intermolecular base-paired domain forms and splicing occurs.
An exa~ple of this second type of responsiYe molecule is illustrated in ~ig. 2. ~his molecule has an intron located between th~ riboso~e recognition seguence and the initiation codon of a single protein-coding region which encodes a desired polypeptide. Thi~ intron prevents translation because it placeæ the ribosome recognition s~quence too far away from the initiation codon. In this example, the intron i8 a self-3plicing intron derived from the pre-rRNA of TetrahYmena. Introns of this type can fold into a structure which causes two cleavage reactions~
one on either side of the intron, and a ligation reaction which joins the portions of the RNA molecule flanking the intron. An essential step in the self-splicing of such introns is hybridization of a region of the intron,~
referred to as the 5'-splice junction, to a second region of the intron, referred to as an internal guide sequence.
Thus, one way the self-splicing activity of the intron can be regulated ifi by preventing hybridization of the~ 5'-splice junction to the internal guide sequence. The responsive RNA molecule depicted in Fig. 2 has - a regulatory domain which is distinct from the intron and the protein-coding region. This regulatory domain has an inhibitor region which is complementary to the substrate region which in this molecule includes the 5'-splice junction of the self-sp~icing intron. Intramolecular base ~13~6~3 W093/23532 ' PCT/US93/04240 ~ -pair formation between the inhibitor region and the substrate region prevents hybridization of the 5'-splice junction to the internal guide sequence, and splicing is prevented. This responsive RNA molecule is designed so that the inhibitor region is also complementary to the anti-inhibitor region of the signal nucleic acid. Thus, in the presence of the signal nucleic acid, the inhibitor region hybridizes to the anti-inhibitor region freeing the 5'-splice junction for participation in the self-splicing reaction.
Referring to Fig. 2 ! respon~ive RNA molecule 40 ha~
a 5'-end 42, and a 3'-end 44. Adjacent to 5'-end 42 is a leader region 49 adjacent to which is a self-splicing intron 48, and then polypeptide-encoding exon 50.
Regulatory domain 46 lies within }eader region 49. Self-splicing intron 48 thus lie~ betveen regulatory domain 46 and -Yon 50, and i8 flanked on its 5' ~ide by a ribosome recognition seguence 56, and on its 3' side by an AUG
codon 66. An inhibitor region 52 within regulatory domain 46 is comp}ementary to a substrate region 54 at the junction between lead r region 49 and self-splicing intron 48. Within~the regulatory domain, on the 3'-side of the --inhibitor region, is a nucleation- region 45 which is contiguous with the inhibitor region 52 and comple~entary to a region of the ~ignal nucleic acid immediately adjacent- to the anti-inhibitor region referrad to as the anti-inhibitor extension. Th- règulatory region may al~o include an inverted repeat 47 on the 5'-side of the inhibitor region. ' Substrate region 54 includes ribosome recognition seguence 56, 'a 5'-splice ~unction 58, and a - stabilizer region 60. - S~lf-spIicing intron 48 is overlapped by a 5'-splice-~'junction 58, and a 3'-splice junction 64 adjacent to AUG codon 66, and includes an internal guide sequence 62.
Referring to Fig. 2A, when inhibitor region 52 hybridizes ~o substrate region 54 a base-paired aomain 70 ~orms preventing 5'-spllce junction 58 ~rom interacting W093/23s3~ ~ 1 3 5 ~ g 3 PCT/~S93/~4~0 with internal guide equence 62. The inverted repeat can fold so as to create a stabilizer hairpin 63.
In the presence of a signal nucleic acid, an intermolecular ba~e-paired domain forms between the anti-S inhi~itor and anti-inhibitor extension regions of the signal nucleic acid and the inhibitor and nucleation regions of the responæive RNA molecule. This interaction frees 5' splice junction 58 allowing it to interact with internal guide sequence 62. Under the~e circu~stances, a ~elf-~plicing reac~ion OCCUr6 . Thus, referring to Fig .
2B, signal nucleic acid 71 having a 3'-end 72 and a 5'-end 73 includes an anti-inhibitor region 74 and an anti-inhibito~ extension 77 which hybridize to inhibitor region 52 and nu~leation region 45 forming ba~-paired domain 75.
The self-splicing reaction remo~es all of the self-splicing intron. The spliced molecule now can produce the encoded polypeptide from exon 50 becau~e the ribosome recognition ~equence is now in clo~e juxtapos~tion to the initiation codon of the polypeptide encoding exon allowing utilization of the initiation sequence as the first codon of a polypeptide.
Referring to Fig. 2C, spliced molecule ga includes 5'-end 42, 3'-end 44, leader region 49, exon 50, ribosome --recognition se~uence 56, initiation codon 66, and fused splice junction 95 containing a small portion of 5' splice junction-58 and a small portion of 3I splice junction 6-4.-- -Any intron known to have self-splicing ac~ivity can be adapted for use as a responsive RNA molecule. Suitable self-splicing RNA can be derived form the nuclear pre-rRNA--of Tetrahvmena, the mitochondrial pre-rRNA of Saccharomyces and NeurosDor~, the introns of ~rao~cteriu~
or Azoarc~s, and the mitochondrial pre-mRNA of Saccharom~ces or other equiva~nt group I self-splicing RNAs. Group II introns can also be us~d in this invention, or any RNA which has at least RNA cleavage activity. RNA ligase activity can be provided by other RNA molecules or their equivalent~

~ 1 3 ~
W093/23s32 : PCT/US93/0424~ ~

Once a self-splicing ~NA has been selected it must be correctly po6itioned between the riboso~e recognition sequence site and the start codon of the polypeptide encoded so that after the self-splicing reaction has S occurred the ribo~ome recognition sequence is positioned correctly relative to the start codon. In eucaryotes translation generally begins at the most 5' AUG of a capped RNA providing that the sequence surrounding the AUG
conforms to A~GNN~ÇG. Accordingly, the responsive RNA
molecule must be designed 80 that this sequence appears only after splicing has occurred. Moreover, an AUG or other codon in a favorable se~uence context can be included in the intron so that it is recognized and used as the 5' most translation initiation site. The inhibitory effect of this upstream AUG on translation initiation at the downstream site will be relieved only upon removal of the intron by self-splicing, thus ~nsuring that no scanning ribosomal subunits reach the downstream initiation site from which tran~lation of the toxic protein would occur.
In a variation on thi~ type of responsive RNA
molecule the self-splicing-intron is placed so as to interrupt a polypeptide-coding--sequence. As illustrated in Fig. 2D, this molecule has an intron located between two exons that together encode the desired polypeptide.
If the intron includes a stop codo~ translation will be blocked. Even if the intron does not encode a stop codon, tran~lation of the intron may be out-of-frame with the downstream exon and/or will a~d- amino acids to the 3Q polypeptide that will likely destroy activity. Removal of the intron results in the fusion of the two exons and formation of a translatable nuc-leotide sequence coding for a polypeptide having the desired activity.
Referring to Fig. 2D,-responsive RNA molecule-40 has a 5'-end 42 and a 3'-end 44. The polypeptide is encoded in two regions, 50 and 51, separated by self-splicing intron 48. Intron 48 is overlapped by a 5'-splice W093/23532 ~ 3 a ~ ~ 3 PCT/US93tO4240 junction 58, and a 3'-splice junction 64 and includes internal guide &equence 62. The protein-coding region SO
is preceded by a ribosome recognition sequence 56 and a translational initiation codon 66. An inhibitor region 52 lies within exon 50 and is complementary to substrate region 54 which overlaps th~ 3'-end of region 50 and the 5'-splice junction 58 and includes stabilizer region 60.
Flanking the inhibitor region on its 5' side is nucleation region 4S that is contiguous with the inhibitor region and is co~plementary to regions in the signal ~ucleic acid immediately adjacent to the anti-inhibi~or region.
Referring to Fig. 2E, when the i~hibitor region 52 hybridizes to substrate region 54 a base-paired do~ain 70 for~s and thus prevents the 5'-splice junction 58 from interaeting with the internal ~uide sequ~nce 62.
Referring to Fig. 2F, signal nucleic acid 71 having a 3'-end 72 and a 5'-and 73 and including an anti-inhibitor region 74 and an anti-inhibitor extension 77 hybridizes to the inhibitor region 52 and nucleation resion 45. The intermolecular base-paired domain 75 is formed. Under the e circum~tances, the 5'-splice junction 58 is free to interact with the internal guide sequence-62-and ~elf-splicing occurs.
Referring to Fig. 2G, the ~elf- plicing reaction remove~ all of the self-splicing intron 48 leaving the fus~d spliced junction 95 which contains portions of the~ ~-5'-splice junction 58 and the 3'-splice junction 64.
Other strategies, for example, where the substrate and/or inhibitor regions are contained within the intron, may be used so that upon splicing these elements are completely removed. When the substrate or inhibitor _~
domains remain in the protein-coding regions, their sequences must be carefully chosen to pre~erve the biological activity o~ the encoded protein. -The degeneracy of ~he genetic code, the possibility of guanosine-uridine base-pairs and conservative amino acid changes that do not eliminate the protein's activity will ~ 1 3 ~
W O 93/23532 - PCT/US93/04240 ;

all be considered. Moreover, it i8 known that many proteins contain regions not essential to their inherent activity and that amino acid changes and/or additions in these areas do not result in a drastic loss of biological S activity. The placement of the substrate and/or inhibitor domains in such a region simplifies the choice of the anti-inhibitor containing ~ignal RNA since change~ to the protein-coding sequence might be more easily tolerated.
The requirement that the inhibitor region be complementary to both the anti-inhibitor region and the substrate region places certain constraints on the sequences of these regions. First, as noted above, the substrate region does not have to have the sa~e sequence as the anti-inhibitor region of the signal nucleic acid.
Since the anti-inhibitor region can be selected but not aItered, the anti-inhibitor region must include a sequence identical to the sequence of the 5'-splice junction. The : minL~al S'-splice junction in a ~ 3~LD~gl_ rRNA intron is only f nucleotides lon~. Since any four nucleotide sequence should occur with a probability of 1/64, many potential anti-inhibitor regions will include the sequence ' of the 5'-splice junction. It -is very likely that many different four-base seguences-can-~serve as a 5'-splice junction provided that the sequence of the internal guide : : 25 reg~on is adjusted to acco~modate the changes in the 5'-~: ~plice junction (Zaug et al.,~' Nature.324:430, 1986).
While it is suitable for the minimal S'-splice junction to be able to base pair with the internal guide sequence, a complex with a single mis-match can-be functional ~Zaug et .

aI, Biochemistrv 27:8924, 19881..--...
The base-paired domain formed by hybridization of the :~
inhibitor region and the substr,ate region must be more ~:
:: stable than the base-pairing that occurs between the 5'- ' :,-splice junction and the internal guide seguence during a splicing reaction. This can be accomplished by choosing an inhibitor region and substrate region that will .:
hybridize to ~orm a base-p~lred domain longer than that W093/23532 ~ 1 3 ~ 6 ~ 3 PCT/US93/04240 formed by hybridization of 5'-splice junction to the internal guide fiequence. The ~ubstrate region is designed to include a stabilizer region that extends the homology between the substrate region and the inhibitor region S sequence beyond the 5'-splice junction. This stabilizer region can be located ju6t 3' of the 5'-splice junction in the case of self-splicing introns located ketween the ri~osome recognition equence and the initiation codon.
This arrangement insures that the stabilizer domain will be removed as part of the splicing reaction and will not interfere with the relationship betw~en the ribosome reco~nition sequence and the in~tiation codon. The ribosome recognition sequence can also be included within the region which base-pairs w~th the inhibitor region, but there is no reguirement that this be the ca~. In the case of a ~elf-splicing intron which is in~erted between exons which encode portions of the same polypeptide, the stabilizer rQgion should preferably be located within the intron, i.e., on the 3'-~ide of the 5'-splice junction ~o that it will be r~moved along with the re~t of the intron~
It is important that the inhibitor/substrate base-paired domain be disrupted only by the signal nucleic acid and not by other nucleic acids present in cell. As- --discusced above, for the first type of re~pon~ive RNA
molecule, this means that the intramolecular base-pair ~ormation must be extensive enough to be disrupted only by a unique nucleic acid. This requirement can make it difficult for the signal nucleic acid to disrupt the intramolecular duplex. As outlined above, the lnclusion of a nucleation region adjacent to the inhibitor region will favor hybridization of the inhibitor region to the-anti-inhibitor region. --Many arrangements of the regulatory dom~in o~ theself-~plicing intron and the exon will be useful. -As noted above, the self-splicing intron can be located between two exons; under these circumstances while the most 5' exon of the unspliced molecule can be translated ;~13' Gg3 W093/23~32 -i PCT/US93/04240 a complete functional polypeptide cannot be produced. The inhibitor region can be located on the 5'- or the 3'- side of the self-splicing intron or possibly within the intron itself. Since RNA is synthesized in the 5' to 3' direction, it is preferred to locate the inhibitor region on the s~-side ~o that the inhibitor will be synthe~ized and have an opportunity to hybridize to the 5'-splice junction before the production of the internal guide sequence~ The inhibitor could be located on the 3'-side of the self-splicing intron if folding of the RNA to form the splicing complex is-slow compared to rate of synthesis of the inhibitor region.
It is preferred that the self-splicing reaction be specific and accurate; if the splice occurs at the wrong location, the ribosome binding site will be positioned incorrectly. In the case of a self-splicing intron located between two exons, incorrect splicing may result in an out-of-frame fusion of the polypeptide encoding sequence~. Self- plicing introns in which the distance between the internal guide ~equence and the 5' -splice junction is relatively short tend to catalyze more accurate splicing reactions. -It- is also important to insure that there are no sequences that will be recognized as al~ernative 5'-splice junctions.
The above described respon~ive RNA molecules can be prepared by any standard methodology. For example, the-RNA can be produced by a transcription of a DNA molecule, either ~n vivo or ~ vitro. Generally, the RNA molecule will be produced by construction of a plasmid or viral DNA
w~ich includes sequences encoding the responsive molacule, appropriate sequences for regulated transcription of the responsive RNA molecule, and appropriate sequences for replication of the DNA. In constructing the RN~ molecule, the general considerations are described above. From a practical viewpoint, it is generally preferred to identify an appropriate RNA molecule having enzymatic activity which is able to cleave itself or other RNA molecules and ,, , ,., . , .. . , .... . . ~ .. .. ~ . .

W093~23532 ~ 1 3 ~ ~ ~ 3 PCT/US93/04~0 is preferably able to 6plice those two RNA molecules together, ç.~., a self-6plicing RNA molecule. The DNA
encoding this RNA molecule is then modified to change the encoded 5'-splice junction and the internal guide seguence as required within the li~itations described above so that the encoded 5~-æplice junction i6 comple~entary to part of the inhibitor region of the responsive RNA molecule. The transcribed RNA molecule i6 then caused to be ligated to RNA which encodes the desired polypeptide and to RNA which includes an appropriate regulatory domain. If required, nucleation site~ and inverted repeats can be designed into the regulatory domain.
The experiments discu~sed in the following Examples 3-7 describe preparation of re~ponsive RNA molecules containing inactive intron~ ~which c-n be reactivated by the presence of specific signal RNAs. The re~ponsive RNA
molecules were prepared fro~ the self-splicing intron or intervening sequence (IVS) in the rRNA of Iee~rn~En~
For the IVS to self-splice requires the ~ 20~ proper folding of the core structure of the IVS RNA.
- ~ IncIuded in this requ~red conformation i8 a base-paired region Xnown a~ P(l) that encompasses the 5'-splice site (Fig. 3). In P(l), th~ internal guide sequence in the IVS
base nairs with the adjacent portion of the 5' exon to for~ a stabl- ste~-loop ~tructure. The 5'-6plice sit~
loc~ted within this stem. The ability of the IVS RNA to self-splice relies on the ability o~ the P(l) stem to form.
A natural ~eguence just upstr~am of the 5'-splice ~30 site can also form a hairpin structure with the exon ~equence immediately ad~acent to the 5'-splice site (Fig.
3). The stem-loop required for ~elf-splicing, P(l), and this alternative stem-loop, termed P(-l), are mutually exclusi~e since the 5' exon sequence immediately ad~acent to the splice site is included in both structures. The alternative stem-loop structure, P(-l), can be made more stable by extending its stem region. See Woodson and Wog3/23~32~ l 3 5 6 ~ 3 ; PCT/US93fO4 Cech, Biochemistrv, 30:2042, 1991, reporting results of a one-nucleotide change in the 5' exon (A to C change at position -14 relative to the 5'-splice site). In that mutant, self-splicing was reported to be decreased.
Conversely, RNAs containing mutations in the 5' exon which either dimini~hed the relative strength of P(-l) or abolished it completely reportedly showed an increase in celf-splicing activity. Three mutants which contain sequence changes in the 5' exon, which were predicted to strengthen the alternative structure, P(-1), were made.
In all three mutants, the level of ~n vitro self-splicing (as judged by the formation of ligated exons) ~as decreased relative to a parent construct in which the natural 5' exon seguence is pres~ent. one ~utant, in which the stem of P(-l) has been lengthened by 5 additional base-pairs, exhibits no detectable self-splicing activity }~ vitro. ! ;- ~
Applicant demonstrated thst self-splicing activity can be recovered even in this strong, non-splicing mutant by the addition of signal RNAs complementary to the upstream 5' exon sequence (inhibitor region) involved in the alternative structure. By binding to the 5' portion of the P(-l) stem, these signal RNAs-disrupted P(-l) and left the s-quence immediately adjacent to the 5'-splice ~ite in single-stranded form, fully capable of hybridizing to the internal guide ~equence in an ~c~tive,~~self-splicing conformation containing P(l). -~ ~
!
Exam~le 3: Plasmid Construction and DNA Dreparation T~e source of-the IVS-containing fragment used to prep~re the responsive RNA molecules was pla~mid pTTlA3T7 (obtainod from Dr. A. Zaug; eguiva-lent such plasmids are readily constructed and this plasmid is used only for purpo~es of illustration of the invention), which contains the 482-bp ~h~I fragment of Tetrahvmena thermo~hila rDNA
incerted into the ~i~dIII site of pT7-2 (U.S. Biochemical Corporation, Cleveland, Ohio) on ~i~dIII linkers. This ~ 13 S ~ 9 3 W093/23~32 PCT/US93/0~240 fragment contains rDNA sequence corresponding to 32 nt of 5' exon, the 413 nt IVS, and 37 nt of 3'-exon. The ~indIII fragment of pTTlA3T7 was isolated and in~erted into the ~indIII site of pTZ19R (United States Biochemical Corporation, Cleveland, OH) to generate a plasmid containing the IVS and a small portion of the natural rDNA
sequence in~erted into-the first few codons of the lacZ' gene, the ~-complementation fragment of the ~-galactosida6e gene. It ha6 b~en reported previously by others (Been and Cech, ÇÇll 47:207, lg86; Price and Cech, Science 228:719, 1985; Waring et al., Cell 40:371, 19%5), that ~-galactosidase activity in E. coli relies on the ability of the IVS RNA to excise itself and ligate the lacZ' coding region in frame so ~8 to produce a translatable mRNA product. ~a vitro mutagenesis was ried out on the pTZ19R derivative containing the rDNA
insert to generate a clone in which the corresponding lacZ' RNA would ~elf-6plice and ~aintain the correct reading frame. -In addition, a potentially u6eful ~
site wa~ created in the 3'-exon and an in-frame AUG in the 3'-exon was destroyed to insure that it not be used as a translation start site. The final DNA ~equence and -correct reading frame of the 3'-exon ~rom the 3'-splice site (-) to the ~iadIII site (underlined) in the vector seguence i8 shown below.
pTETBLU ~ -- -T AAG GTA GCC AGC CGT CGA CAT CTA ATT AGT GAC GCa,aÇ

pTETB~U DNA was then used as the parent for a series -- -of splicing mutants in which changes were made by in vit~o mutagenesis in the 5' exon sequence to improve the base- _ pairing ability in the alternative P(-1) stem-loop structure. Care was taken to maintain the correct reading ~rame in the spliced RNA product and to a~oid the creation of translational start or stop codons. The resulting sequence changes made in the 5' exon RNA and the RNA

~ 1 3 ~ 6 1 ~
W093/23~32 PCT/US93/04~0 alternative structures predicted to form are ~hown in Fig. 3.
All site-specific mutations were generated using the in vitro Mutagenesis Kit from United States Biochemical Corporation. DNA oligonucleotides were made on an Applied Biosyste~s 394 DNA/RNA Synthesizer using phosphoramidite chemi~try and purified using OLIGOCLEANr columns (United State~ Biochemical Corporation) prior to use as mutagenic oligonucleotide. Plasmids were maintained in strain MVllgO (E. coli ~ Isrl-recA~ 306::TN10 ~ (lac-prol thi-su~E rF' ~ro A+B~ lacIQ lacZ Mi5 traD36). Each plasmid was veri~ied by DNA equencing ~Tabor and Richardson, Proc. Natl. Acad. ~ci. USA 84:4767, 1987). --Plaomids for UB~ as~n vitro transcription templates -~
.
- 15 wer- purif~ed by Qiagen (Qiagen Inc., Chatsworth, CA) ; raxi-colu~n preparation as descrhbed by the manufacturer except that the final DNA preparation (400 ~1) was extracted two times with an equal volume of phenol, once with ohloroform, and~ethanol precipitated in th presence of 0.25 ~-Tris-HCl, pH ?.~5. The plasmids were linearized by ~cleavage with either ~SQRI or ~mHI to generate t~plates ~on which runoff T7 transcription will yield ùll-l~ngth RNA of~548 or 527 nt,--r~spec~iv ly. ; (The T7 ;pro~oter ~s-quence is located i ~-diately upstream of the - 25 polycloning site and within the coding sequ~nce of ~-galactosidase.) Example 4: Sianal RNAs Short signal ~NA~ (11-26 nt~ were chemically synth-~ized on an Applied Biosy~tems 380B DNA synthesizer ~30 using phosphoramidite chemistry. Prior to u~e, the signal RNAs were desalted u~ing a C18 SEP-PAC~ cartridge (Millipore Corporation), gel-purified and quantified by -~ absorbance at 260 nm. Signal RNAs were stored at -20C in 1 mM EDTA, 10 mM Tri~-HCl (pH 7.5). The fieqUences of the cignal RNAs specific for precursor RNA from PTET1419 and pTET21-24 (see FIG. 3) are given below:

W093/2353' ~l 3 ~ 6 ~ 3 PCT/US93~04~0 pTET1419 4S 3' GCCGCUCUCAG. 5' 4S3 3' GCCGCUCUCAGUGAU S' pTET21-24 8S4 3' CGcccAuuuAAAuc~c~çAçyGAuA 5' 12S 3' CGGAAACGCCCAU W AAAUCUCUCAG 5' The~e ~ignal RNAs are co~plementary to the upstream exon sequence which form~ the 5' side of the P(-1) ste~ in the given construct. The underlined nucleotides correspond to the portion of the signal sequence that will ba~_ pair with 5' exon sequence involved in ~he P(-1) stem, the remaining nucleotides ba~e pair either with nucleotides at the base of the ~tem or in the loop. Por example, signal RNA 4S3 will base pair with 4 nt 5' to the base of the stem in pTET1419 RNA, all the nucleotides included in the 5' side of the P~-1) stem and 3 nucleotides in the loop.
In pTET14 RNA (~ee FIG. 3),~a U to C change at -14 relative to the 5'-~plice site.allow~ the for~ation of an extra C-G base-pair to lengthen the P(-1) stem. This particular 6equence change wa~ reported by Woodson and Cech (Woodson and Cech, Biochemistrv 30:2042, 1991) to decrease self-splicing activity of a short precursor RNA.
pTET1419 RNA has an additional nucleotide change (G to A
at -19) which allows P(-l) to form a. more stable stem by . creating an A-U base pair in place of a less stable G-U
base pair. Finally, pTET21-24 RNA ha~ a very stable P(-l) stem generated by 4 additional nucleotide changes (a~
positions -21 to -24 relative to the splice sit~). ~
Calcul~ted free energies at 37C for these structures, based on the most current values in the literature (Freier .-et al., Proc. Natl. Acad. Sci~ USA 83:9373, 1986; Jaeger et al., proc. Natl. Ac~d. Sci. USA 86:7706, 1989), are _ !
~lso giYen in Fig. 3. In all of these constructs, ~ ~
nucleotide changes were made in the upstream 5' exon only, without altering the TVS or the ~3 nt at the 3' end o~ the 5' exon.
On templates linearized with ~ç_RI or ~HI, full-length transcription from the T7 promoter yielded ~13~6~
Wos3/23~32 PCT/US93/~240 transcripts of 548 and 527 nt, respectively. These differed only in the length of their 3'-exon (92 vs. 71 nt), but had equi~alent lenath 5' exon6 (43 nt) and IVS
RNA (413 nt). Correct ligation of the 3'-exon to the 5' exon with excision of the IVS yielded an RNA of 135 nt for the EcoRI runoff tran~cript and 114 nt for the corresponding E~H~ transcript. The appearance of ligated exons ie an indication of the level of self-splicing supported by a particular IVS-containing construct. ~ ~
:.:
Exam~le 5: Decreasina Self-Splicina b~ Increasina Stability of P(-1~.
I~ vitro transcription was performed as follows.
Transcription reactions using T7 RNA polymera~e were carried out in transcription buffer (40 ~M Tris-HCl, pH
15 7 . 5, 5 rM MgCl2, 10 mM dithiothreitol, 4 mM spermidine) containing 500 ~M each NTP and -10 ~i t~32P]CTP.
Individual reactions (10 ~1 total vo}ume) contained 0.1 ~g linearized plasmid temp}ate and 20-30 U T7 RNA poly~erase.
After 30 minutes at 30C, 2 ~l of each sample was removed 20~ and~mixed wit 2 ~l buffered for~amide containing xylene cyanol FF ~and bromphenol blue (formamide/dye~ mix). The remainder of the sampl- was w~r~ d to-37C, and 2 ~l of 1 M NaCl, 20 mM MgC12, 1 ~M GTP was added to adjust the reaction conditions to better support~splicing. After 15 or 60 minutes as noted, 2.5 ~1 samplès were removed and mixed with 2.5 ~l of the formamide/dye m-ix. Samples were anallyzed on denaturing gels containing 4% (19~
acrylamide:bisacryla~ide and 7M urea in 0.4 X TBE (TBE is -89 ~M Tris, 8~ mM boric acid, 0.025 mM EDTA).
Electrophoresis was carried out at_30-60 watts using 0.4 X TBE as running buffer. Gels were exposed to ~odak XONAT
XAR-5 film.
~ For gel purification of 32P-labelled, precursor RNAs, transcription reactions were scaled up 2.5- to 10-fold and incubated 1-2 hours at 37C. In some cases, the concentration of each NTP was increa6ed to 2~5-3 mM in an wo 93/23532 h 1 3 ~ 6 4 ~ PCT/US93/04240 attempt to reduce self-splicing during the transcription reaction and thereby maximize the recovery of full-length tran~cripts. An equal volume of formamide/dye~ was added to the completed reaction and the entire reaction was loaded onto a denaturing gel as described above. After visualization by autoradiogr~phy, the region of the gel containing the full-length transcript was excised and placed in 0.5-1 ml 0.5 M ammonium acetate, 1 mM EDTA.
A~ter 12-16 hours at 4C, the eluent was removed and the RNA precipitated by the addition of 2.5 volumes of ethanol. The final RNA pellet was resuspended in 1 mM
EDTA, 10 mN Tris-HCl (pH 7.5) and stored at -20C.
Transcription using the parent plasmid and the modified constructs as template~ was casried out in the presence of t~32P~CTP to generate 32P-labelled transcripts that could be analyzed for their abil~ty to self-~plice (Fig. 4, 0 minJ. Full-length transcripts (FL), a slight amount of IVS RNA (IVS), and additional ~intermediate" RNA
products (*j, were pre~ent for all template~. A small amount of an RNA product of the appropriate length to be ligated exons (LE) from the ~çoRI run-off transcript (135 nt) as well as from the E~E~I run-off transcript (114 nt) -was also visible, and indicated that a limited amount of~
splicing could occur under these transcription conditions.
This faint band decreased in intensity with the order pTETBLU>pTET14>pTET1419 and was not visible in pTET21-24. -- -From analysis of the resultant RNA products, it is-clear that transcription of the parent plasmid, pTETBLU, generated transcripts cap~ble of efficient self-~plicing. - --This i8 evidenced by ~n increased amount of ligated exons 15 and 60 minutes after adjusting the conditions to better --_-support splicing. -~
By comparison of the amount of ligated exon produced,it is apparent that transcripts from pTET14 and pTET1419 were still capable of self-splicing, although less efficiently than transcripts from the parent pTETBLU.
Both pTET14 and pTET1419 produced fewer ligated exo~s than -~ ~ 3 5 ~ 4 ~ PCT/US93/Q4240 pTETBLU when shifted to splicing conditions, and of thQse two mutants, pTET1419 was the least efficient. Under the same condition~, however, transcripts from pTET21-24 d~d not appear to self-splice. No ligated exons were visible for pTET21-24 precursors after conditions were altered to support splicing. The relative observed ability of these three mutant con~tructs to self-splice, then, follows the order expected based on the increasing stability of the P(-l) ~tem, i~, there i~ a negative correlation between the strength of the P(-l) stem and the RNA's ability to self-splice. Noreover, the presence of the highly stabilized P(-1) stem in pTET21-24 reduced ~n vitro splicing to undetectable levels.
Under splicing conditions, a number of RNA products in addition to the ligated exons were visualized. As expQcted, splicing of the~pTETBLU tran~cript generated a sign~ficant a~ount of the excised IVS RNA in its various forms (circular and linear IVS and the shortened forms lacking the 5' 15 or 19 nt). Some of these product~ were visible for the ~utant transcripts às well, even for pTET21-24- where no ligated exons were visible. The pre~ence of these IVS products may-reflect the ability of these mutant RNAs, which are to variouF-degrees ~isfolded at the 5'-splice site due to a strong than normal P(-l) ~tem, to still ~upport hydrolysis at their 3'-æplice site (See Woodson and Cech, Biochemistrv- 30:2042, 1991).
Although no released 3'-exon was visible, one RNA product-that was greatly enhanced in the mutant RNA lanes (indicated with an asterisk in--Fig.- 4), waæ of the appropriate ~ize to represent the~ 5! exon-rVS RNA. This 5' exon-IVS RNA would still be _expected to undergo circularization reactions, producing the linear IVS
products (~-15 and ~-19) seen on the gel. The short RNA
indicated with an arrowhead is unidentified. This RNA
increased in intensity after the switch to splicing conditions. It also seemed to increase in abundance as wos3~23~32 ~ 1 ~ S ~ ~ 3 PCT/VS93/04240 the ability of the precursor RNA to self-splice decreased, and thus was ~ost prominent in the pTET21-24 RNA lanes.
It is cl~ar from the lack of ligated ~xon~ in the pTET21-24 lanes that this mutant was unable to undergo correct ligation of the two exon products. The apparent side reactions of the mutant IVS-containing RNAs (e.a., the formation of the RNA product ~abeled with the asterisk) when unable to undergo a correct splicing reaction may be able to be used advantageously. For example, this Hself-destruction" may be beneficial for rVS-containing mRNAs that encode toxins where rapid turnover of the message would further diminish the possibility that a toxin be produced in the absence of the proper signal.
i 15 ~ExamDle 6: Reactivitv of sDlicina Reaction by Sional RNA
Gel-purifi~d, full-l ngth RNA precursors were subjected to ~plicing conditions in the absence or ¦
pre~ence of signal RNAs to test the ability of ~hort RNAs comple~entary to the upstream 5' exon sequence to disrupt .~, 20-~the~ P(-l) structure and thereby allow the active P(l) structure to form.
~- Splicing reactions u6ing~gel-purified precursor RNAs were carried out by incub~ting 0.1-0.25 pmole of 32p_ labelled transcription 10 ~1 spIicing bu~fer (200 mN NaCl, ;
25200 ~ GTP, 30 mM Tris-HCl, pH 7.5) in the presence of 0 ~-~
to 1000-fold molar~exce~s of signal RNAs. After warming to 37C, MgC12 was added to 5 mM to initiate the splicing I -reaction. Incubàtion periods ranged from 10 to ~20 - -minutes at 37C, at which times s~mples were removed and 30 mixed with an egual volume of formamide/dye. Samples were _ - analyzed on denaturing gels as described above. := _ -If self-sp}icing were reactivated, more ligated exon products would be expected to be produced in the presence -of these signal RNAs than in their absence. R-sults of experiments demonstrating reactivation of the splicing wo 93~23s32 ~ ~3`~ 6 ~ ~ PCT/USs3/04240 reaction are given for pTET1419 RNA` in Fig. 5 and for pTET21-24 RNA in Fig. 6.
As seen previously in Fig. ~, incubation of pTET14 19 RNA under splicing conditions in the absence of any signal s RNA generated a small amount of ligated exon product.
With gel-purified transcript, this was again the case (Fig. 5). Tt may be ~hat the P~-1) stem in pTET1419 RNA
is not stable enough to completely inhibit the formation of P(l), 80 a small amount of splicing still occurred.
The amount of ligatæd exons produced increased, however when either of two specific 6ignal RNAs was present in incubation. Even with an extremely low 6ignal-to-tran~cript ratio (0.1:1), a ~light elevation in the amount of ligated exons was seen. As the signal-to-transcript 1~ ratio was increased (up to 100~0:1), the production of igatQa ~xon~ al80 ~increaB-d. The~e experiments showed that-the ability of pTET1419 RNA to correctly self-splice and produce ligated exons responds directly to the pres-nce of a specific signal RNA, and that a significant level of self-splicing is recovered.
A ~imilar respon~e to signal RNAs was ~een with gel-purified pTET21-24 RNA (Fig. 6). As noted before, with pT~21-24~ RNA, no ligated~ exons~ were~ visib}e when the transcript was incubated alone (see al~o Fig. 4). This indicates~ tb~t ~the P(-l) stem in pTET21-24 RNA_ is suff~iciently~ftable to complet ly inhib~it th ior~ation of P(l). Upon addition of either of two signal RNAs (8S4 or 12S) ~peoific for this transcript, however, ligated exons are produced. That th~ 32P-labelled RNA products are ligatQd exons can be seen by comparing their--length to that of ligated exons produced from pTETBLU RNA._-Splicing ... . .
of transcripts produced from EcoRI-digested _templates produced ligated exon~ of 135 nt in length. Transcripts from templates linearized with ~HI produced ligated exons that wére correspondingly shorter (114 nt). Thus, even though the splicing reaction was turned completely -o~" in the pTET21-24 RNA itself, it was still possible ~:, . . .. . . . .

~ 1 3 .'j fi ~ 3 WOg3/23532 PCT~US93~04240 to reactive the splicing reaction with a specific si ~ al RNA.
For the EçoRI runoff tran cripts shown on the left ~f Fig. 6, there was a ~econd major product (indicated with a dot) that also see~ed to respond to th~ pre~ence of the signal RNAs. This RNA is shorter than the correctly ligated exons, and at this tLme it~ origin in unknown.
Splicing at an alternative site or a specific breakdown of-the RNA are possibilities.

Example 7: ~s~ bLl3a~ ssay ~ hen grown on LB or B agar plates containing 5-bromo-4-chloro-3-indoyl-~-D-galacto6ide (X-gal~, a chro~ogenic substrat~ of ~-gal~cto~ida~e, pTETBLU-containing colonies are dark blue a~ expected for a colony producing ~-galactQsidase. Since the coding region of the Q-complem~ntation fragmen~ of ~-galacto~idase on pTETBLU is interrupted by the Tetrahv~ena IVS, this RNA ~ust be correctly ~elf-~plicing in order to produce an active ~-fragment. If self-splicing is not occurring, 5top codons present in all three raading frame~ in the IVS would not allow translation into the downstream portion of the gene.
For comparison, a control plasmid (pTETUIB) in which the intron-containing ~indIII fragment from pTETB~U is inserted into pTZ19R in the reverse orientation was constructed. For this control, where no splicing can occur due to the wrong orientation, the resulting colonies are white.
Theoretically, then, cells containing mutants which are deficient in splicing should produce lighter blue colonies, while colonies of non-splicing mutants would be white. Under standard growth conditions, cells aontaining pTET1419 and pTET21-24 mutants grew as colonies that were considerably lighter in color than cells containing the parent plasmid pTETBLU, but not white. This appears to 3S indicate that even the strongest non-splicing mutant, pTE~21-24 (as judged by its in~bility to or~ ligat~d ~13~64 ~
W093/23~32 PCT/US93J04~0 exons n vitro) is still capable of forming the minimal amount of spliced me 3age necessary to support translation of a le~el of ~n ~-fragment of ~-galactosidase that could confer blue color to the colonies. O~her scientists have noted ~-galactosida~e activity (blue colony color) with IVS-containing onstructs in which self-splicing should have left the ~-galactosidase ~e6.age in an untranclatable frame (Been and Cech, Cell ~:207, 1986; Price ~nd Cech, S~iençe 228:719, 1985). It may be that alternative splice sites exist.
For a ~ore quantitative deter~ination, ~- i galactosidase assays were carried out cn plasmid-containing cell~ growing in cultureO (Niller, ~xperiments in Molecular ~çneti~, Cold spring Harbor Laboratory, Cold Spring ~arbor, N.Y. ~1972). For this as~ay, o-nitrophenyl-~-D-galacto~ide (ONPG3 was used ~s the chromogenic ~ubstrate because its product after cleavage with ~-galacto6id~se can ke mea~ursd spectrophoto- ¦
~etrically. A control pl~smid (pT ~ ) was con~tructed in which the intron-containing ~iadIII fragment from pTETBLU was in~erted into pTZ19R in the reverss orientation and was used to determine background levels of ~pontaneous breakdown of ONPG. In the~e exper-iments,-cells containing either the parent plasmid or the splicing mutants were grown under inducing conditions (}~ç~, in the _ pre~ence o~ IPTG, a lactose analog). Production of active ~-galacto~ida#e in cell containing the pTET1419 and pTET21-24 ~plicing mutants was reduced to a few percent of the parental valu~s, ~hus indicating that the changes in the RNA were reflected, not only by a decrease in the amount of ~ vitro self-splicing, but by a concomitant decrease in the a~ount of active protein produced Ln_the coli cell. Use The respon ive RNA molecules of the invention are useful for producing plant cell that respond to the presence of a given virus. In many instances there is no way to pr~vent viral infection of such cells. The ~13~6~3 W093/23~32 PCT/US93/04240 molecules of the invention will allow creation of plant lines that are resistant to any given ~irus in that any plant cells which become infected will be destroyed before the virus is able to spread to other cells.
This section describes the methods by which a responsive RNA can be u~ed to affect the physiological state or viability of a particular cell type. In the ca~e of re~ponsive RNA ~olecule~ that are regulated by the formation of a base-paired do~ain within a protein-coding region the method requires construction of a responsive RNA which encodes a protein which will affect the physiology or viability of a cell; and identification of an signal RNA which is specific to the cell type, e., an RNA molecule which carries a nucleotide ~equence that is only pre~ent or accessible in the RNA population of the cell type which is to be affected. For responsive RNA
molecules regulated -by self-splicing introns the method requires construction of a re~ponsive RNA which encodes a protein which will affect the physiology or viability of a cell. The active protein must be tran61ated from the spliced message and not the unspliced message. It also requires identification of a signal RNA which i8 specific to the cell type, i~e., an RNA molQcule which carries a nucleotide sequence that i6 only pre6ent or acce sible in the RNA population of the cell type which is to be affected.
For example, a re~pon~ive RNA can be designed to specifically kill: virus-infected plant cells containing viral RNA and not uninfected cells; cells containing mutant RNA and not cells containing wild type RNA; cells in a particular tissue and not other kinds of cell in the plant.
The efficacy of such a responsive RNA in altering the phy iological state of a cell will depend upon the responsive RNA being delivered to the location in the cell where the signal nucleic acid resides; the responsive RNA
having all of the nucleoside sequences required for all ~3~6~
W093/23532 PCTtUS93/04~0 42 =
the processes leading to production of the encoded protein including splicing, poly-A addition, capping, transport acros6 the nucl~ar membrane, and tran~lation initiation;
and the respon~ive RNA may al~o carry sequence elements 5 which confer stability to RNA in the nucleus as well as -the cytoplasm.
A r~ponsive ~NA molecule can be delivered into a - cell in the form of RNA or in the form of a gene made of DNA or RNA. Delivery of RNA into a cell can be accomplished by needle injection, electroporation, polyethyleneglycol precipitation, or by the use of liposomes including tho6e made of cationic lipids.
Delivery of the re~ponsive RNA in the form of a gene can be accomplished by the use of a non~irulent virus or ~5 b~cteriu~. Thi~ would reguire the insertion of the responsi~e RNA-encoding gene along with the transcriptional or replicative ~ignal element~ into the genome of the virus. Retroviru~es, polyo~a viruses, and ~ ~
vaccinia virus have b~en engineered which are capable of ' ~-delivering and expressing genes, and other viruses could be developed and used for this purpose.
Another general method of u~ing a responsive RNA to -control t:he~phy~iology of an organi~m or ~ particular cell type involves a responsive RNA gene integrated into the 25 cellular genome via any plant transformation technique, _ e . a ., A ~ obacterium tumifaciens. The activation of ~ ~ ~-splicing of the responsive RNA could be caused by -exogenously added polynucleotides.
Other e~bodiments are within the following claims. --- ~
. . 1:

I

Claims

Claims 1. A responsive RNA molecule having a ribosome recognition sequence, a regulatory domain, a substrate region, and encoding, in one or more protein-coding regions, a polypeptide; said regulatory domain comprising an inhibitor region complementary to said substrate region; said inhibitor and substrate regions being capable of forming a base-paired domain in the absence of a signal nucleic acid; said base paired domain reducing the level of translation compared to that level observed in the absence of said base-paired domain; said signal nucleic acid having an anti-inhibitory region complementary to said inhibitor region which, when base-paired with said inhibitor region, increases the level of translation of said responsive RNA compared to the level of translation of said responsive RNA observed in the absence of said signal nucleic acid to cause a hypersensitive response in a plant cell; wherein said signal nucleic acid-comprises part of the nucleic acid of a plant cell infecting organ-ism.

2. The responsive RNA of claim 1 wherein said protein-coding region is an exon.

3. The responsive RNA of claim 1 wherein said substrate region comprises part of one said protein-coding region.

4. The responsive RNA of claim 2 wherein said substrate region comprises part of an intron.

5. The responsive RNA of claim 2 wherein said substrate region comprises part of an intron adjacent to the 5'-end of one said exon.

6. The responsive RNA of claim 1 wherein said substrate region includes part of said ribosome recogni-tion sequence.

7. The responsive RNA of claim 6 wherein said ribosome-recognition sequence is a ribosome binding site.

8. The responsive RNA of claim 1 wherein said responsive RNA is purified.

9. The responsive RNA of claim 1 wherein said polypeptide modifies cell viability, cell proliferation, transcription of DNA, translation of RNA, or replication of DNA.

10. The responsive RNA of claim 9 wherein said polypeptide has cytotoxic activity or ribonuclease activity.

11. The responsive RNA of claim 10 wherein-said polypeptide is selected from the group consisting of the active subunit of diphtheria toxin, the active subunit of cholera toxin, ricin, and the hok, gef, RelF or flm gene products of E. coli.

12. The responsive RNA of claim 4 wherein said intron prevents the complete translation of said one or more exons.

13. The responsive RNA of claim 4 wherein said intron reduces the level of translation of said one or more exons compared to the level of translation of said exon in the absence of said intron.

14. The responsive RNA of claim 4 wherein said intron is located between said ribosome recognition sequence and a protein-coding region.

15. The responsive RNA of claim 4 wherein said first intron is located between two said exons.

16. The responsive RNA of claim 4 wherein said intron is bordered at its 5'-end by a 5'-splice junction and at its 3'-end by a 3'-splice junction.

17. The responsive RNA of claim 15 wherein said substrate region comprises a 5'-splice junction bordering said intron.

18. The responsive RNA of claim 16 wherein said intron catalyzes two RNA cleavage reactions, one within said 5'-splice junction and one within said 3'-splice junction.

19. The responsive RNA of claim 18 wherein said substrate region comprises the 5'-splice junction of said intron.

20. The responsive RNA of claim 19 wherein said inhibitor region reduces the level of occurrence of said cleavage reaction within said 5'-splice junction.

21. The responsive RNA of claim 1 wherein said signal nucleic acid is single-stranded.

22. The responsive RNA of claim 10 wherein said signal nucleic acid is a viral RNA.

23. A DNA molecule encoding the responsive RNA
of claim 1.

24. The responsive RNA of claim 19 wherein said responsive RNA comprises a 5'-splice junction RNA of Tetrahymena thermophila having at least one base modified compared to a native 5'-splice junction.

25. A method for specifically interfering with the growth of a plant cell harboring a signal nucleic acid by introducing, into the cell the responsive RNA wherein said responsive RNA comprises a ribosome recognition sequence, a regulatory domain, a substrate region, and encoding, in one or more exons, a polypeptide; said regulatory domain comprising an inhibitor region complementary to said substrate region; said inhibitor and substrate regions being capable of forming a base-paired domain in the absence of a signal nucleic acid; said base-paired domain reducing the level of translation of said responsive RNA molecule compared to the level of translation in the absence of said base-paired domain;
said signal nucleic acid having an anti inhibitor region complementary to said inhibitor region which, when base-paired with said inhibitor region increases the level of translation of said responsive RNA compared to the level of translation of said responsive RNA observed in the absence of said signal nucleic acid to cause a hypersensitive response in a plant cell; wherein said signal nucleic acid comprises part of the nucleic acid of a plant cell infecting organism.

26. The responsive RNA of claim 1 expressed in a transgenic plant.

- 4?/1 -27. A plank comprising the responding RNA of

claim 1.