Note: Descriptions are shown in the official language in which they were submitted.
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
W093/23532 PCT/US93/04
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
14
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 ;
W093/23532 PCT/US93/04240
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
18
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
21
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 ~ -
22
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~ ~
24
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 ;
26
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
29
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
32
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
33
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
37
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
38
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
39
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
