Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 94/29481 21~ ~ 3 2 ~ PCT/US94/06034
DETECTION OF NUCLEIC ACID SEQUENCES
FIELD OF THE INVENTION
The present invention concPrnc a method and kit for the
detection of specific nucleic acid sequence in a sample.
BACKGROUND OF THE INVENTION
DetPcticn of the presence of a specific DNA or RNA sequence
in a sample~is required for a variety of ~ nt~ ostic and
therapeutic purposes, e.g. detection of a specific mutation in a sample of
amniotic fluid, p~ A~e testing~ testing for incorporation of a viral DNA
into aL cell's genomic DNA, etc. The task of direct ~letection of a specific
DNA or RNA sequence, which is routinely pel~lllled by the use of an
a~ropliately labelled probe, is often hindered by the fact that the specific
DNA or RNAis present in a sample only in minute amounts.
Ex~l~lcs of mPt~c which enable the amplification of DNA
sequences present in a sample in only minute qll~ntities are: LCR (ligase
chain reaction), 3SR(self-sllct~ine~ sequence repli~tion) or PCR ~olymer-
ase chain-reaction). In PCR a sample is cQnt~r,te~l with a primer DNA
comrl;...~ y to a 3' end sequence of the specific DNA, a DNApolymer-
ase and with single DNA nucleotides. Following a number of replication
cycles, the sample is enriched with the specific assayed DNA. Atypical
cycle of PCR co.--l.-;ceC ~ree distinct stages: a first stage in which ~e
double-str~n~e~DNAis melted to two single strands; a second stage of
SU~ S~t~!R~
WO 94129481 PCT/US94/06034 . -
~k~20
annealing of the primer to the single-stranded DNA, and a third stage of
poly...L.;~Iion where the annealed primers are e~t~n~ l by the DNA
polymerase, to produce a double-str~nded DNA. The cycle of melting,
~nnP~lin~ and DNA synthesis is repeated many times, the products of one
cycle serving as templates for the next ad thus, each s~lcce~ive cycle
enriches the sample with the specific DNA.
PCR suffers from several shollcolnillgs, the most serious of
which being its lack of specificity. The e~ilive hybridization telll~ldlul~,
i.e. the ~elll~e.dlule in which the two strands of DNA hybridize, ~ s
the specificity of the re~tio~- A low effective hybridization teln~dlul~,
results in a higher ~rce.lldge of non-specific binding. In PCR this
e, which is defined by the telll~dlUl~, of the ~nn~lin~ stage, is
l~,lali~/ely low and this brings about non-specific binding of the probe to ~e
target sequences resllltin~ in amplification of undesired sequences which
brings about a relatively high background reading.
This non-specificity also le.luil~s an ~rlrlition~l and time-
con~l-min~ detection procedure such as ele~L,oplloretic separation of the
amplification products on an agarose gel, in order to s~te b~lween the
various ~mplific~tion products, and does not enable detection of the presence
of the assayed DNA by a mere ~letection of amplifir~tion
PCR also suffers from a severe problem of co~ ;on
which is due to ampli~c~ticn of sequences ~at did not origin~te from the
test sample being seq~Pnces ~ ion~lly introduced to the sample.
Another disadvantage of PCR is that it is a complex procedure.
Typically, each of the stages of melting, ~nnP~ling and polymPri~tion is
carried out at a different te~ el~ , e.g. melting at 94C, ~nn~2~1inp at
50C and poly.... ;~ n at 72C. Since the s~.l~les have to be co~t~ntly
cycled through seve~al lell~ lu~ a special ap~ u~ is required ren~prin~
the procedure laborious and time con.~-lming
SU~ J~ ~tt~l (R~LE 26)
WO 94/29481 216 4 3 2 3 ; ; PCT/US94/06034
Another shortcoming of PCR is in the time required therefor.
A typical cycle lasts several minlltes, and usually 25-30 cycles are required
to produce sufficient copies of amplified DNA. Thus, a typical PCR even
in a completely automated system lasts at least 2 to 3 hours.
Finally, PCR is basically suited for the detection of DNA
sequ~nces. Where detection of RNA sequences is desired, RNA has to be
converted first to DNA (by reverse transcription). This conversion to DNA
l~Ui1~3 ~ itif)n~l time, effort and ~ yllles, and also introduces many errors
due to the inherent in~ccllracy of reverse transcription.
It should be noted that although PCR is advantageous in
obtaining large amounts of a specific DNA, such as for producing large
qll~nlitiee of probes for genetic assays, it is often an "over-kill" where
merely the presence of a specific DNA sequence in a sample is to be
assayed.
Other such m.o.ths)-ls such as 3SR (WO PCT 89/05631) and
Target Nucleic Acid Amplification/Detection (WO PCT 89/05533) are
relatively rapid isothPrm~l plocesses for DNA letectiQn However, these
mPths--le also suffer from lel~live;ly effective low hybri~li7~tion te~ s
which are even lower than those of PCR, typically in the range of 37~1C.
These low le~ alule3 drastically reduce the specificity of the procedure
due to non-speçific probe-target binding, and in cases of çlinir~ gnostics,
this may result in an intolerable level of mie~ gnosie.
Additionally, amplification str~tegies such as Target Nucleic
Acid ~mplifir~ti~ n/Detection that are based on the amplification ~l~.lLies
of a replicase-type en_yme are unreliable due to the possibility of spontane-
ous RNA amplification in the absence of target (Chetverin-AB, et al., J.
Mol. Biol., 222(1), 3-9 (1991)).
It is the object of the invention to provide a method for the
detection of a nucleic acid sequence which is:
SUB~ ul~ Shtt~ (R~LE 26)
WO 94129481 PCT/US94/06034 . ~ -
~6~3~
(i) reliable and sequence specific due to the minim~ tion of
incorrect target-probe hybridization;
(ii)i relatively rapid;
(iii) ec~l*~ ly ico~ ,-ic elimin~ting the need for specialized and
expensive a~a dlus;
(iv) .~laLi~ely simple, not requiring the ~ ition of a large number
of dirr~elll enzymes or nucleotide pools; and
(v) ~m~n~ble to ~ n by enabling the amplification process
itself to be in-lic~tive of the presence or absence of the nucleic
acid sequence to be assayed.
Further objects of the invention will become clear from the
following description.
GLOSSARY:
Below are the me~ning,C of some of the terms which will be
used in the following description and claims. For ease of reference, ~e
reader is also referred to the acco"l~ljing drawings (the numbers in
bracl~tc in the Glossary below refer to the item numbers in the drawings):
Assayed nucleic acid sequence (ln?~?n?~02,402,50?~6Q?~1402) The DNA
or RNA sequence which presence in the sample is to be ~letecte~l
First DNA molecule (2?Q ~0,4?Q ~?û"~F?Q 142O) - a DNA molecule having
a double-stranded, i.e. functional promoter and a 5' end sequence which is
complem~nt~ry to ~e 5' end portion of the assayed nucleic acid sequence
(102, ... etc.).
Second DNA molecule (222,322,432,57?~Ç?-?~1422) - a DNA molecule
comprising a single-stranded 3' end sequence being complement~ry to the
3' end portion of ~e &~ed nucleic acid seyu~,lce (102, ... etc.) and fur~er
comprising a sequence which can be tr~n~rihe~ to ~e triggering
SUBSTIl UTE SHEET (RULE 2B)
WO 94/29481 PCT/US94/06034
2~6 432~
RNA sequence (see below). The 3' end sequence of the second DNA
molecule and the 5' end sequence of the first DNA molecule may be
complPmpnt~ry to the entire assayed nucleic acid sequence or to only a part
~ereof, leaving an int~rmf~i~ry portion in ~e assayed nucleic acid sequence
having no complemPnt~ry COulltC~ in either the first or second DNA
molecules.
Thirdl DNA molecule (623) - a single-stranded DNA molecule comple-
mentary to the intPrmediary portion of the assayed nucleic acid sequence.
Detection ensemble (104,204) - an en~Pmhle of molecules comprising ~e
first DNA molecule (220, ... etc.), the second DNA molecule (222, ... etc.),
and where the S' end sequence and the 3' end sequence of the first and
second DNA molecule, le~e~ /ely, are complemPnt~ry together to only a
portioin of the assayed nucleic acid sequence also col..l.. ;~;..~ the third DNAmrll^cllle (623). The ~letectinn en~ernhle optionally compri~e~ also a ligase.
In the presence of the assayed DNA (102, ... etc.) and the transcription
reagents (see below) the detection en~emhle is activated and an RNA
tr~n~crirt (110,210,310,410,510,610,710,810,910,1010,1110,1210,1310,
1410,1510,1610) co~ ;sing the triggering RNA sequence (see below) is
produced.
T~ggering RNA sequence - a sequence in ~e RNA l.. ~ l (110, .. etc.)
transcribed from the second DNA molecule (222, etc.), which is only
produced after activation of the detection en~emhle. This RNA sequence is
then capable of tri~erin~ transcription in the RNA am~lification ensemble
(see below) of a signal RNA molecule (see below) co...l~ the signal
RNA sequence (see below).
Tl ;66~. ;ng RNA molecule (110,210,310,410,510,610,710,810,910,1010,
1110,1210,1310,1410,1510,1610) - the RNA molecule compri.~in~ ~e
tri~erin~ RNA sequence.
SUBSTIl~l E SHEET (RULE 2B)
WO 94/29481 PCT/US94/06034 .
~1~432~
Signal RNA sequence - a seqllPnce in the transcription product of the RNA
amplification ensemble (see below). The production of the signal RNA
sequence, above a baseline level, indicates the presence of the assayed
nucleic acid sequence in the sample.
Signal RNA molecule (116,716,816,916,1016,1116,1216) - the RNA
molecule comprising the signal RNA sequence.
Signal DNA sequence - a DNA sP~llPnre serving as a template from which
the signal RNA sequence is transcribed.
Transcription reagents (113,213,413,513,713,813,913,1013,1113,1213,
1313,1413,1513,1613) - RNA polymerase with single RNA nucleotides and
buffers required for RNA transcription.
Transc~ption system - a DNA homoduplex or a DNA/RNA heteroduplex
c. ~ a filncti~n~l promoter and a d~w~ DNA or RNA sequence
which can be transcribed upon activation of the promoter into an RNA
transcript.
RNA amplification ensemble (Figs. 7-13, 15,16) - an en~mhle compri~in~
ç~nti~lly the tri~erin~ RNA molecule (110, ... etc.) and the transcription
re~Pnt~ (213, ... etc.) in the first embo-limPnt of the invention; or the
triggering RNA molecule (116, ... etc.), the tr~n~srirtion re~ent~ (213, ...
etc.) and fourth DNA molecule (see below) in the second embo~limpnt; or
the triggering RNA, the transcription re~gentc and afifth and sixth DNA
molecules (see below), in the third embo~limPnt of ~e invention; or the
trj~;~rin~ RNA, the tr~n~erirtion reagents, and a seventh and eighth DNA
molecule in the four~ embo~1im~nt of the invention. The RNA amplifica-
tion ~ le opticn~lly co~ ,s a ligase. In one emborlimpnt of the first
emboclimPnt, the presence of the trjg~ering RNA together with the
tr~n~ription system is sufficient for the transcription of the signal RNA
sequence. In the second embo~limPnt ~e tri~ering RNA hybridizes with
the fourth DNA molecule. In the third embo~limPnt, the tri~ering RNA
SU~ (R~LE 28)
WO 94/29481 ~ 16 4 3 2 ~ PCT/US94/06034
hybricli7es with the fifth and sixth DNA molecules brin in~ them together.
In the fourth emborlim~nt the triggering RNA hybridizes with the seventh
or eighth DNA molecules. The RNA/DNA hybrid produced in accordance
with the secon~ mird and fourth embo-lim~nte, serves as a template for the
production of the signal RNA sequence.
Fourth DNA molecule - a DNA molecule (1148) which is part of the RNA
amplification eneemhle in acco~allce with the second embo~limPnt This
molecule c~....l,.;ees a promoter which is single-stranded in at least an
es~en~i~l part thereof and is thus inactive. It further comprises the signal
DNA sequence. When the triggering RNA sequence, which in ~is
embodiment is complennlont~ry to the single-stranded part of the promoter,
hybridlizes with the single-stranded part of the promoter of the fourth DNA
molecule, a functional promoter is produced and thus the signal RNA
molecule can be transcribed.
Fifth DNA molecule - a molç~lle (12S2) which is part of the amplification
eneennhle in accold~lce with the third embo~limPnt It co...l.. ;~e~ a
functional promoter, and at its S' end, a single-stranded sequence which is
complem~nt~ry to the 5' end portion of the tri~gerin~ RNA sequence.
Sixth DNA molecule - a molecule (1259) which is part of the amplification
ensemble in accordance with the third embo-lim~nt It comrri.ees at its 3'
end a single~ e~l sequence which is complem~nt~ry to the le ..~;..;..g 3'
end portion of the tri~gerin~ RNA sequence and in addition, comrriees the
signal DNA sequence.
Seventh DNA molecule - a moleclllr (1580) which is part of the amplifica-
tion Pn~en ble in accordal-ce with the fourth embo-limPnt It has a
fimrtion~l, double-stranded promoter, either a priori pr~aled or ~ee~mhled
from two single stranded seqll~n~ec, linked to an ~ntie~nee sequence
cQmrlem~ nt~ly to the 3' end se~ re of the tri~gering RNA. One or a few
end nucleotides in the 5' end of the template strand of this molecule
SU~ lt SH~ (R~L~ 26)
WO 94/29481 PCT/US94/06034 ~ -
2IG432Q 8
could be RNA nucleotides. The 3' end sequence of the triggering RNA
hybridizes to said ~nticPn.ce sequence and after ligation the promoter can
induce RNA kanscription, the triggering RNA serving as a template.
Eighth DNA molecule - a molecule (1529') which is part of the amplifica-
tion PncPmhle in accor~ce with the fourth emboflimpnt It is similar to the
seventh DNA molecule, the di~.~.lce being in the ~ ;c~ce sequence which
in the eighth DNA molecule is identical to the 5' end sequence of the
kiggering RNA molecule. The transcription product of the seventh DNA
molecule/triggering RNA hybrid can thus hybridize to the eighth molecule
and the so formed hybrid serves there as a tPmpl~ for ~n~c~iption of RNA
molecule having the seq~l~nce of the trig~ing RNA seqllence, which in turn
can activate again the seventh DNA molecule in a "ping-pong" ...~ P .
Promoter molecule - a DNA molecule (1429) which is part of the dPtection
ensemble according to the fourth embo-limPnt of the invention and is
PssPnti~lly identical to the eighth DNA molecule.
Adapter molecule - a DNA molecule (1431) comprising a sequence
complçmPnt~ y to the non-template sequence immediately ~-ljnGPnt the
promoter sequence in ~e promoter molecule possibly having one or a few
RNA nucleotides at its 3' end.
Joiner molecule - a DNA molecule (1433) co...~ at its 5' end a
sequence which is complPlnPnt~ry to a 5' end portion of the adapter
molecule and a se4ucl~ce in the le~ g 3' portion of the molecule which
is co~ lf ..~ to 3' end portion of the first molecule in accoldallce with
the fourth embo~lim~nt The joiner molecule serves for joining the first and
the adapter molecule in the fourth embo-iimPnt
DNA iniation sequence (I)IS) - a DNA seqllen~e present d~wl~ of the
promoter of the seventh or eighth DNA molee~ s which enhances transcrip-
tion of the seqllpnre present duw~ n thclc~ln, by the RNA polymerase.
SUBSI~ Shttl (R~LE26)
WO 94/29481 PCT/US94/06034 -
~164320
,' s ~ r ~ '~
SUMMARY OF THE INVENTION
The present invention is based on a novel concept for the
detection of a nucleic acid sequence present in a sample (assayed nucleic
acid). The present invention is useful for the detection of a nucleic acid
sequence which may be DNA or RNA having a known sequence. The
method is based on a system co"~p-;~in~ two components: a detection
en~çmhle and an RNA amplification~n~emhle.
If the assayed nucleic acid sequence is present in the sample,
the detection ~n~emble gives rise to the production of a triggering RNA
seq~nr~ The 1ri~g~in~ RNA sequence can initiate an RNA amplification
reaction in the RNA amplification çn~en~ble, to produce a signal RNA
sequence, which can then be detecte~l by means known per se. If the
assayed DNA sequence is not present in the sample, the triggering RNA
sequence and consequently the signal RNA sequence are not pro-11ued
Thus, the presence or absence of the signal RNA sequence in~ tes the
presence or absence, respectively, of the assayed DNA sequence in the
sampIe.
As can be seen, the presence of the assayed sequence is only
required for the first step, in the initial ~letection en~Pmble and is no longerre.luL.cd for the RNA amplification ~n~em~le. This uncoupling of the
detection and amplification steps allows for various ~ lAtion~ of the
letecti- n ~ r. . .hle such as A~ iti~ n of blocker molecules and the raising of
alul~s, which reduces non-specific hybri-li7Ation. For example, after
hyhri-ii7Atic)n, the l~ r ,~I...c may be raised once to a tempc~dLul~, in whichall non-srecific hybrides will melt and only the specific hybrid will remain.
All said ~ Ati~n~ have to be carried out only once, during the detection
SIJ~ UI~ S~IEE~ (R~LE 2&)
WO 94/29481 PCT/US94/06034 .
2 ~ o
stage, and the cycles of amplification do not require any i~ ../elllion and
can thus be easily automized.
Furthrrmore, th¢ detection of the presence of the assayed
nucleic acid seq l~nre results in RNA amplification which may for example,
be detecte~l by light absorbance at a frequency of 260 nm, with no need to
open the reaction vessel and separate the various amplification products.
The detection of RNA amplification per se obviating the need to ~ e
whether a specific amplification product is present in the re~ction llli~Lu~e
c-n~;~lerably simplifies the whole process as well as elimin~tes co..~
tion of other reaction vessels by aerosol particles from the assayed reaction
vessel which is a current problem enco~lleled in other amplific~tion
procedures.
In accordance with the pl~3_ 1l invention, there is no need to
amplify the assayed DNA sequence, but rather its presence brings about
production of large ~ l;es of the signal RNA sequence. The method of
the invention thus involves prod~lcti~n and ~mrlific~tion of RNA rather than
amplifir~ti()n of DNA as in PCR C~o~ 1y there is no need for meltin~
of the two DNA strands duling amplifir~tion cycles since ~e RNA removal
from its template occurs during the normal course of transcription, and
accordingly the repetitive tem~ dlul~e cycles of PCR are avoided.
Another advantage of ~e meth-)cl of the invention is that,
collll~y to DNA replir~tion, in RNA tr~n~r~irtion several RNA polymerase
enzymes can operate on the same template ~imlllt~neously and thus the
overall transcription process is relatively rapid. Furthermore, the speed of
RNA production can be increased if the RNA molecule pro~ Ge(l in the
amplification P.n~çmble and which col"~ es the signal RNA sequence,
compri.~es also ~e triggering RNA sequence itself, which triggering
sequence can in turn activate additional transcription in a self-amplifying
SU~ ul~Sl~tt~ (R~IE26)
wo g4ng48l 216 4 3 2 0 PCT/US94/06034 .
11 ' '
m~nnP.r, and thereby the proch~ction of RNA advances exponf nti~lly in a
very rapid m~nner.
- - Thus, the method of the invention provides a relatively
speciiEic, rapid and uncomplicated method for the detection of an assayed
nucleic acid sequence in a sample.
The present invention provides a method for detçctin~ the
presemce of an assayed nucleic acid sequence in a sample, comprising ~e
steps of:
(a) re~ctin~ the sample with a f-1etection çn.~f Inhle co~ g
- a first DNA molecule having a promoter sequence and a 5'
end sequence which is co...~ mf~nt~ry to the 5' end portion of
the assayed nucleic acid sequence;
- a second DNA molecule co..~l.,;.~il-g a single-stranded 3' end
sequence being complf-~mf~nt~ry to a 3' end portion of the
assayed nucleic acid sequence, and further colnrri~in~ a
sequence which can be transcribed into a tri~rin~ RNA
sequence capable of initi~ting a re~rtion in an a~r~,;ate
transcription system in which an RNA molecule having a
signal RNA seq~f~nr~ is being prof-l-lcef1 the 3' end sequence
of the second DNA molecule and the 5' end sequence of the
first DNA molecule may be complc ..f -~In.~/ to the entire
a~yt;d nucleic acid seql)f nr~ or to only a part ~ereof leaving
an ;"1~ .".eA;;~ portion in the assayed nucleic acid having no
complf~ment~ry counl~L in either the first or the second
DNA molec~lf,s, in which case the detection en.~en-hle fur~er
- colll~l;ses
a third DNA sequence being complf mf~nt~ry to said intrrme-
diate portion;
SUB~ ultSHttl (2~LE26)
WO 94/29481 PCT/US94/06034 .
2 ~
12
(b) incl-b~tin~ under conditions to allow hybridization of said first DNA
molecule and said second DNA molecule and were present also said
third DNA molecule with said assayed nucleic acid sequence, and
optionally adding a ligase to allow ligation of adjacent ends of said
first, second and third DNA molecules;
(c) adding transcription re~ nte comprising an RNA polymerase and
RNA nucleotides and inc~lb~tin~ under conditions to allow the
fonn~tion of RNA transcripts having said triggering RNA sequence;
(d) cont~r,tin~ the RNA t~necrirts with an RNA amplification er~eernhle
in which the trigg~ring RNA sequence in(lllce~s fo/."nlion of RNA
molecules co.,~ the signal RNA sequence; and
(e) ~tectin~ the presence of said signal RNA sequence, positive results
in(lir~ting the presence of said assayed nucleic acid sequence in said
sample.
Said first molecule may co- "l" ;~e a double-stranded and hence
fi-nction~l promoter. ~ltrrn~tively, the promoter is a priori single-str~n~ed
in at least an e~ee~nti~l part thereof and a sequence complement~ry to the
single-str~n~led portion of the promoter is added during or after step (b).
Namely, it should be lmtl~r.etood that by the above definition of first DNA
molecule, the functional promoter may be present a priori or may be
~eeemhled in sihc during the pc rv..,.n..ce of the assay in the assay vessel.
Steps (a) and (b) of the method of the invention may be
modified to increase the specificity of the detection andlor prevent
production of short sequences of RNA transcribed from the first DNA
moleclll~, which may increase the background signal. These modifications
include, for example, an additional step after step (b) of raising the
tempcl~aLul~ to a point where only p~.lf~illy m~tçhed hybrids of assayed
nucleic acid seqll~nree and first and second DNA molecules (and also third
DNA molecule if present) remain hybridized while all other hybrids in
SD~lJIul~S~ (R~LE26)
WO 94/2g481 216 4~ 2 Q PCT/US94/06034
which the individual strands do not perfectly match one another are melted.
The reformation of micmAt(l-ed hybrids after melting can be p~ lled by
the addition of blocker molecules which compete with the assayed nucleic
sequence by hybri~li7in~ at a high affinity to the first or to the second DNA
molecules. Such a modification ensures that triggering RNA is produced
only in case of a perfect match beLweell the assayed nucleic acid sequence
and the first and second DNA molecules.
In order to avoid prod~lction of undesired short RNA
tr~ncçrirts from the first DNA molecule which would have an effect of
increasing assay "noise ", it is possible to Accçmhle the promoter of the first
DNA molecules in stages. In this case, the first molecule comrricec a
promoter which is single-str~n~le~l in at least an çcc~ntiAl part thereof and
thus non-functicn~l After the form~tion of hybrids of the assayed nucleic
acid sequence and the first and second DNA molecules, blocker DNA or
RNA molecules are added which hybridize only to the ~ee first DNA
molecules in such a m~nn~r so as to avoid subsequent hybri-li7~tion thereto
of the mi~in~ promoter part and cannot hybridize to first DNA molecules
present in the hybrid. A DNA molecule co...~ g the mi~sin~ promoter
part is then added, which co~ lctes only the promoter of first DNA
molecule in said hybrid rPn~lering it ffinction~l and thus enabling the
production of the tri~rin~ RNA. In co~ sL to this, the molecule
~ ~e mi~sin~ promoter is unable to hybridize with free first DNA
molecules which are blocked, and thus no short RNA ll~lscl;~L~ are
produced from free first DNA molecllhs.
Free first DNA molecules can also be se~A. ~ from hybrids
of assayed nucleotides and first and second DNA molecules, for example,
by h~ving the second DNA molecules bound to a solid support, e.g.,
m~ netic beads and thus, after hybridization, removing all non-bound, i.e.
free, DNA molecules.
SU~ ult S~t~ (R~LE 26)
WO 94/29481 PCT/US94/06034.
~4~
14
If the production of short RNA transcripts from the sample is
avoided, it is possible to detect the presence of the assayed nucleic acid
seqll~n~e by ~etecting the mere amplification of RNA per se, for example,
by a change in the absorbance, with no need for ~e detection of the
presence of a specific signal sequence.
Finally, it is possible to detPrmine whether the only transcript
produced is a short transcript transcribed from the first DNA molecule or
whether in aMition, triggering RNA is produced by using synthetic,
nucleotides labeled with a fluorescent moiety. One type of these non-
n~tllr~lly occllrring nucleotides which are recognized by the RNA polymer-
ase, may be introduced in the coding region first DNA molecule, and a
second type may be introduced in the second DNA molecule. The synthetic
nucleotides complementary to those present in the first DNA molecllle are
labeled with one type of fluorescent, i.e. yellow. The synthetic nucleotides
c~mplem~nt~ry to these present in the second DNA molecule are labeled by
another type of fluorescc~-l, i.e. blue. After the reaction takes place, the free
synthetic nucleotides are separated from the reaction lllixlule, for example
by washing through a cl~a~ged filter through which the neutral synthetic
nucleotides pass. If only short RNA transcripts from the first DNA
molecule are produced the fluorçscçnce in ~e reaction nlixlwe will be
totally yellow. If tri~in~ RNA tr~n~ ed from both the first and second
RNA molecules is produced the fluorescence will be both eyllow and blue
and will be seen as a green color.
In acco~ ce with a first embo~limPnt of the present invention,
the tri~ring RNA sequence col.~p,;.~es a sequence which can bring about,
.,lly or by hykri~ in~ to various DNA moler~ s, the production of self-
repli~ ~ting RNA, namely an RNA which serves as a template for form~tion
of i-l~ntir~l RNA molecules by an RNA poly.,.~ ~e Examples of such self-
replif ~ting RNA's are X-RNA and Y-RNA (~on~r.~
S~ ts~ttl ~R0lE26)
~ WO 94/29481 216 ~ ~ 2 0 PCT/US94/06034
Sher, P.A., Cell, ~!~3), 608-18, (1990). In accordance with the first
embo~impnt~ the RNA amplification en~çmhle, comprises RNA nucleotides
and am RNA polymerase and at times DNA molecules which are required
in some embo~limPnt~ for ~cs~mhling a functional promoter. The signal
RNA which is produced in the RNA amplification Pn~emhle is a self-
replicating RNA. The presence of self-replicating RNA in the sample, in
acc~ ce with the first embo~limPnt, indicates the presence of the assayed
DNA sequence in the sample.
In accordance with a second embc-limPnt of the l)resent
invention, the amplification en~çmhle comrri~eC a fourth DNA molecule,
which co~ çs a promoter which is single-stranded in at least an e ssçnti~l
part ~hereof, and is thus inactive, and further co",l.,;ces a signal DNA
seqllPnre which is ~ ;hed into ~e signal RNA sequence. In accordance
with ~lis embo~limpnt~ the trip;~Prin~ RNA se~lPn~ e is complemPnt~ry to the
ec~Pnt~ single-str~n~le(l portion of the promoter. Wh_n the trj~Prinp RNA
sequence is cont~ctecl with the fourth DNA molecule, the two molecules
hybridize whereby the promoter becomes double-str~n~le~l and thus
functional. Consequently, in the presence of a tr~n~rrirtion system, i.e.
RNA polymerase and RNA nucleotides, an RNA molecule incl~ling the
signal RNA sequence is l~ cl;he~l Preferably, the RNA molecule thus
produced which inçl~ e~ ~e signal RNA sequence includes also the
tri~çrin~ RNA se~lçnre and consequently the process is self-amplifying,
namely, the RNA transcripts pro~lllce-l themselves induce by them~elves
transcription of additional RNA tr~n~crirts.
In accordallce with a third embo-1iment of the invention, the
amplification çn~mhle co~ lises a fifth DNA molecule which has a
fim~til~n~l promoter and a single-stranded sequence at its 5' end and
comprises a sixth DNA molecule which has a 3' single-stranded end
sequence. The trj~rin~ RNA sequence is complçm~nt~ry to the above-
SUBSr~lUrE SHEET (RULE 2~
wo 94/29481 PCT/US94/06034 ~
~1~4~2~
16
mentioned two single-stranded sequences, its 5' end sequence is comple-
mentary to the 5' end sequence of the fifth DNA molecule and its rem~ining
3' end seql~nce is complemrnt~ry to the 3' end sequence of the sixth DNA
molecule. Consequently, the triggering RNA sequence after hybridizing to
the single-str~nde(l sequence of the fifth DNA molecule and the sixth DNA
molecule, brings the respective 5' and 3' ends of the fifth and sixth DNA
molecules together whereupon they can be ligated by the use of the ligase.
In the presence of an RNA tr~n~ee~rirtion system an RNA transcript
a signal RNA sequence is produced. Sirnilarly as in the second
emborlim~nt the fifth DNA molecule optionally co...l..;~e a sequence which
tr~ne~ribes into the triggering RNA sequence and the RNA transcript
produced thcl~Lu~ll serves also as a trigger for pro~ur,tion of additional such
transcripts.
By a morlifi~tion of the ~ird embotlin~Pnt ~e six~ DNA
molecule serves as a template for transcription of a self-replicating RNA,
such as the X-RNA. Once a self-replicating RNA is produced, it serves as
a template for production of further self-replicating RNA molecules.
In accor~ ce with a fou~th embo-lim~nt of the invention, the
amplification ~n~çmhle COlll~l;SCS a seventh and an eighth DNA molecule
both of which have a functional, double-stranded promoter. The promoters
may be, a priori double-stranded or may be ~çmhled in steps from a
single-str~n~led promoter. The seven~ DNA molecule has an ~nti~çn~e
sequence attached to the non template strand of the promoter which is
complemrnt~ry to the 3' end sequence of ~e tri~rin~ RNA. The eighth
DNA molecule has an ~ e seql~en~e attached to the promoter which is
idrntir,~l to the 5' end seqllrnre of the tri~ n~? RNA. When the trj~rin~
RNA is c~nt~Gted with the amplific~tion çn~ç~nhle in accoldal~ce with the
fourth embo~lim~nt~ the RNA hybridizes to the short ~nti~çn~e sequence in
the seventh DNA molecule and after ligation ~e functional
S~ ul~ Snt~ ~R~I~ 2S)
~ WO 94/29481 216 4 3 2 0 PCT/US94/06034.
promoter can induce production of RNA, wherein the triggering RNA can
serve as template. The RNA transcript thus produced can in turn hybridize
in a similar m~nner to the eighth DNA molecule and the RNA transcript
which is produced there is identical to the triggering RNA and can again
activate the seventh DNA molecule. Consequently there is a c~ Qus
cross-tri~g~in~ of RNA transcription and a large number of copies of both
the h^ig~in~ RNA and the ~ntieen~e RNA thereto are produced, which can
both serve as the signal RNA.
If desired, it is possible to construct a promoter made of a
single DNA strand which is able to loop and form a double-stranded part
only lmder proper c~n-liti~n~. When the promoter is not ligated to the RNA
the loop and hence the double-stranded part is not foml~d. Only
when the promoter is ligated to the RNA transcript the loop structure is
stabilized and a double-stranded, functional promoter is formP!~l
In accordal~ce with a slight modification of the fourth
embodiment, it has been found that it is preferable to insert after the
promoter a short DNA initiation sequence (DIS) which is recognized at a
high affinity by the RNA poly~ se and this increases considerably the
~n~rirtion rate of the RNA molecule (~illi~n et al., NAR, ~, pp. 8783
(1987)). One variant of DIS termed nDlSI " should be inserted after the
promoter of the seventh molecule and alwlL~ variant of DIS termed "DIS2 "
should be added after the promoter of the eighth molecule. Since the DIS
is added after the prnmnter~ it becomes ~r~n~cribe~l to the RNA molecule in
each cycle of amplification so that the transcribed RNA molecules becomes
~d~ lly enriched with DISl or DIS2 on both ends and then the amplifica-
tion process is stopped after two cycles. In order to avoid this leng~h~nin~
phenom~n~ a rybozyme, which is an RNA sequence re~ g catalytic
p.~p~lies and capal~le of recogni7in~ and cutting a specific RNA seq~lPnce~,
is inkoduced to the reaction mixture. One type of rybozyme specifically
S~lll~lt SHEET ~R~lE 26)
WO 94/2g481 PCT/US94/06034
~1~43~
18
cuts out the RNA sequences transcribed from DISl and another rybozyme
-specifically cuts out DIS2 after each amplification cycle. The sequence
transcribed from either DIS, termed herein~flcer "disl " and "dis2" may be
cut immediately after transcription when they are on the S' end, or before
hybridization of the triggering RNA to its cognate promoter when it is on
the 3'. The latter option has some adv~nt~s. First higher fidelity at the
3' end. Since the 3' fidelity of the polymerase is not high, the cutting off
the 3' end of the transcript ç~ tes this problem. Another advantage
resides in the fact that cutting by the rybozyme becomes a prerequisite for
ligation which e"sules the correct sequence of events.
In addition to the advantages stated above, the use of
rybozymes in ~-nPr~l offers several adv~nt~gec: first it enables higher
specificy. The recognition sequence of the rybozyme can be made highly
specific so that after each arnplifiG~tion cycle the specificity of the RNA
molecule is verified, thereby reducing background noise due to co~
tion of the reaction by undesired nucleic acid molecllles.
Second, rybo;~yll-cs can replace the need for a ligase. Some
rybozymes are able to specifically ligate two RNA molecules together, so
that the nick that occurs a~er the tri~g~ing t~n~rlipt ~nn~ to its promoter
can be ~ai,~,d by the rybozyme instead of enzymatically.
Third, i~ vv~dread-out~ ~es. There is awide repertoire
of ,~ o~yl~,e e~y~nalic activies. Some rybozymes are able to add a single
mlt~1eoti-1e to the RNA se4~ ce and some are able to cut a single nucleotide
therefrom, for example, during the lig~tion procedure. By using labeled
nucleti~s these plo~c.lies of the rybozymes can be used to ease detection
of the transcription products.
The present invention also provides a kit for carrying out the
method of ~e invention. The kit typically compri~es the various lDNA
SUBSIl~ul~Sht~ (R~LE26)
WO 94/29481 216 ~ 3~ 0 . !` ` ' PCT/US94/06034 .
19
molecules, reagent ~y~ s, etc. required for car~ing it out. Separate kits
for each of the above-mentior~ed embo~limPntc are provided.
The invention will now be illustrated with reference to some
non-limitin~ specific embo-limPnt~ described in the following with
occasional reference to the ~nnPxe~l drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a flow chart of the method of the invention;
Fig. 2 shows the basic components of the (letectioll en~ernhle;
Figs. 3-6 show some modifications of the basic embodiment shown
in Fig. 1;
Fig. 7 shows an amplification en~Pmhle in accordance with the first
embo~imP.nt
Figs. 8-10 show some mn-1ifi~ti-)n~ of the amplification Pn~çmhle in
accordance with the first embo~1imPnt;
Fig. 11 shows an amplification en.~emhle in accordance with the
second embo-l;.. ~;
Fig. 12 shows an ~mrlifi~ti-)n ~ le in acco~l~lce wi~ the ~ird
embo~imP.nt
Fig. 13 shows a modification of the embo-liment of Fig. 12;
Fig. 14 shows a ~letecti~ n Pn~çmhle in accordal~ce with the fourth
embo-1imP!nt
Fig. 15 shows an amplification en~Pmhle in accorda.lce with ~e
fourth embo-limP-nt and
Fig. 16 shows a modification of the embo-limPnt of Fig. 15.
In the drawings, various symbols are used which in the context
of the present description have the following mP~nin~s:
Straight line ( ) - DNA strand
Wavy line (- -) - RNA strand
SUBSTITUTE SHEET (RULE 2B)
WO 94/29481 ~ 1 ~ 4 3 ~ ~ PCT/US94/06034 .
A,B,C, etc. ............... - sequences in the coding strand of a
DNA
A',B',C', etc. ............ - complem~nt~ry sequences in non-
coding DNA strands
a',b'c', etc. - sequences in RNA ~n~crihed fromDNA sequences A,B,C, etc.
a,b,c, etc. ............... - sequences in RNA complelnçnt~ry
to a',b',c', etc.
A",B" - sequences in DNA which are par-
tially complemPn~ry to DNA se-
quences A and B
TRIG - tri~rin~ DNA sequence
t~g hj~gp~rin~ RNA sequence
SIG - signal DNA sequence
sig - signal RNA sequence
p+ fi-nr,tion~l promoter (DNA)P~ - non-functional promoter (DNA)
p~' - functional promoter (RNA)
p~ - non-functional promoter (RNA)
A-a,B-~,A'-a ',B'-,B ',C-y etc. .. - complPnlPnt~ry sequences on the
same strand of nucleic acid seque-
nce
DIS- - DNA initiation sequence
In the figures, the various components are desigr-ed by three
or four digit m-mPr~l~ The first digit in a case of a three digit numeral and
the first two digits in the case of a four digit nllmer~l l~resent the figure
number and the last two digits l~lesent the coll~.llent number. In all
figures like components have the same component number. Thus, for
SUBSTIlVTE SHEET (RIJLE 283
WO 94/29481 ~ ~ 6 4 3 2 0 PCT/US94tO6034 .
example, a component 852 in Fig. 8 has the same function as 1052 in Fig.
10, etc. ~ .
. . i .
METHOD OVERVIEW
Reference is first made to ~ig. 1 showing an overview of the
method of the present invention. Nucleic acid seq~lPnce 102 which is in this
example a DNA sequence, forming part of a ~nome of an or~ni~m in an
assayed sample, is cont~ctçd with a detection en~Pmble 104, comrri~in~
various DNA molecules (as will be elaborated fur~er below) to form a first
reaction mixture 106. The reaction mk~lule is sub3ected to conditions
allowing a hybridization of the assayed sequence 102 with co~ sponding
sequemces in the DNA molecules of the ~letection çn~Pmble (see further
below).
Following hybrirli7~tio~ and optional ligation steps, a
"~ inn system 108 is ob~inp~rl c~ ;..P a DNA heteroduplex having
a double~ P~ i.e. filn~tinn~l promoter and a duw,~ll~" DNA sequence
which can be transcribed into an RNA molecule 110 which is ler~l~d to
herein as the triggering RNA rnolecule. In order to obtain the trjg~Prin~
RNA molecule 110, !~ .C.~ lion re~ntc 113 comprising RNA polymer~e
and simgle RNA nucleotides are added to the ll~lscl;~tion system 108.
The triggPring RNA molecule is then colnl~ined with an
amplification .on~emble 112 to yield a second reaction ll~ 114. The
amplifilcation en~Pnnhle compri~es re~gPnt~ which in the presence of the
trl~gPrin~ RNA seqllPnce will give rise to the production of large quantities
of an RNA molecule 116, rcfc.l~d to herein as the signal RNA molecule.
The detection of the presence of the signal RNA molecule can then be
carried out by any number of means known per se.
SUBSl~lul~Shttl (R~L~26~
WO 94/29481 PCT/US94/06034
2~ 32~ 22
In the following, various fe~lu,~s of the invention will be
described with reference to some specific embo(liment~. Figs. 3-6 and 14
describe various embo-liment~ and modifications of the detection en~çmhle
shown in Fig. 2. Figs. 7-13 and 15, 16 show various embor1iment.c of the
amplification ~n~emhle and the production of the signal RNA molecule.
DETECTION ENSEMBLE
Reference is first made to Fig. 2 showing the basic ~ealules of
the first step in the ~ . . "~-ce of the mPthocl of the invention in which the
tri~ering RNA sequence is produced. From here on ~e invention will be
described with reference to the embo-1imPnt~ in which the assayed nucleic
acid seq~len~ e in a DNA se~ e and it is to be ulld~lood that the method
is applicable also to RNA seq ~Pnce~ rnutatis mutandis.
The detection en~çmhle 204 co.l~ çs a first DNA mole-
cule 220 and a second DNA molecule 222. The first DNA molecule 220
comprises a functional, double-stranded promoter P+. The first DNA
molecule 220 has a single-stranded sequence A and the second DNA
molecule has a single-stranded sequence B linked to a triggering sequence
TRIG which may be single or doul~le-str~n-led The sequences A and B are
complPmPn~ry to se~lue,.ces A' and B', le~e~;lively, in the assayed
DNA 202.
If the assayed DNA 202 is present in the sample, and
a~pr~l;ate con-1itiQn~ for hybridization are provided, a hybrid 224 is
produced. In this hybrid the 3' end of sequence B and the 5' end of
sequence A are ~ cPnt to one another and are optionally ligated to yield
ligation product 226.
Tl2."~ lion reagents 213 co,..~ ing RNA polymerase such
as the T7 poly"le,~se and RNA nucleotides and buffers are then added and
u~tShttl (R~E26)
wo s4ns4sl 216 4 3 2 0 pcTluss4lo6o34
as a result a triggering RNA molecule 210 having a triggering sequence -
trig linked to sequence b' and a'- is produced.
Reference is now made to Fig. 3 showing a modification of the
method outlined in Fig. 2 intPn~led to elimin~te almost entirely the
possibility of o~lai~ g a positive result in case of an h.l~clrect match
bcLwt;~ll the dete~tion en~Pn-hle and the assayed DNA. The right-hand side
of Fig. 3 shows the case of a perfect match between ~e first 320 and
second 322 DNA molecules and the assayed DNA 302; and the left-hand
side of the figure shows the case of an illl~.re~ match, where the assayed
DNA302'c~ r..;~ se~ cesA" and B" (the mi~m~ç~ being repres~nte~
sc~em~tically by loops in sequence A and sequence B").
After hybri~1i7~ti~n b~,Lwtxin the DNAseqllPncç,s, as described
in c~....P~ n with lFig. 2, the l~ -nl-..~ is raised to a te.llper~lur~ wherein
there will be a total melfin~ of the DNA sequences in case of an il~.re.iL
match and less than total mPltin~, e.g 50%, in case of a perfect match. This
l~lll~cl~Lul~ depPnfl~ as l~own, on a .. h.-~ of factors incll-~lin~ the length
of the DNA sequences as well as the relative proportion of the nucleotide
bases A and T versus G and C, and has to be ~ ....;..ed ~ln~il;cally in each
case.
At this t~ ~alulc short DNA fr~gr~n~ e 370 and 372 having
se~ s A' and B', r~ccLi~ely, are added which hybridize to the single-
str~n~lPr1 A and B ~eql~Pn~es. The Le~ ~dLulc is then lc v~er~d and a ligase
and transcription re~nt~ are then added. In the case of an hllp~lre-;L
match, only small RNA tr~n~crirts 373 with the sequence a' will be
produced whereas in the case of a perfect match, an RNA transcript 310
having the ~iggering RNA sequence - t~g will be produced.
In case there is a s~ ifir~nt diCr~ ce between meltin~
~ )~..dLulcS of the above two hybrids, for example, where the hybrid in the
case of any imperfect match beLween the first DNA molecule and the
SUBSTITUTE SHEET (RULE 28~
WO 94/29481 ~ :li 6 4 ~ ~ ~ PCT/US94/06034
24
assayed DNA has a melting temp~lur., Tl which is higher than melting
temp(,alule T2 of the hybrid co"l~ i"~ the second DNA molecule, the
method may proceed as follows: addition of first DNA molecule 320 and
second DNA molecule 322; addition of blocker molecule 370; raising
tempcralule to Tl; lowering te.ll~e.alu,e to T2; addition of blocker
molecule 372; lowering tempe,dlule to reaction temperalule; addition of
transcription reagents 313.
In order to ensure that the blocker molecules 370 and 372 have
an advantage over the "~;~",;.1~ 1-P(1 assayed DNA in re-hybril1i7~tion with thefirst and second DNA molecule when the te~ cl~l~le is lowered, these
blocker molecules should be in excess to the assayed DNA. In addition, it
is possible to add an extra ollJil.~ sequence to first molecule 320 and to
add a sequence comple~nt~ry to said extra sequence, to blocker
molecule 370. This extra sequence raises the affinity b~tweell ~e blocker
molccllle and the f~rst DNA molecule to be higher than the affinity between
the first DNA molecule and the mi.cm~trlled portion of the assayed DNA.
An extra c,.l,iL.~y sequence can be added in a similar m~nner, to second
DNA molecule and blocker molecule 372 re~eclivGly.
R~ nce is now made to Fig. 4 showing a m~ific~tion in the
method outlined in Fig. 2 and 3 which elimin~tes the production of short
RNA ~ t having the sequence a', which are tr~n~t ribed from the first
DNA molecule. First molecule 420 is i~lPnti~l to first molecule 220 in
Fig. 2. Second mrlcc ll~ 422' is e~ lly i-l~ti-~l to second molecule 222
in Fig. 2 and is linked to a m~etic bead 418 at its 5' t~rmin~l. Assayed
DNA 402 is added to produce hybrid 424 optionally followed by lig~tin~ to
yield ligation product 426. Magnetic force is ~en applied. All molecules
linked to a magnetic bead, namely, free second DNA molecules 422 and
li~qtion product 426, are drawn to the magnet 419, while molecules l-nlinke~l
to m~etic beads, namely, first DNA molecules 420 and assay
SUBST~TUrE SHEET (RULE 2B)
WO 94/2g481 PCT/US94/06034 .
~ 64320
DNA molecules 402 are w~lled away. Transcription reagents 413 are added
to the test vessel and since the only DNA molecules cs..l~ a promoter
in the reaction mi~ e are ligation product 426, the only RNA transcripts
which are produced are the tri~erinp: RNA molecules 410 c~ the
trig sequence. This modification enables detection of the presence of
æsayed DNA by the detection of mere amplification of RNA with no need
to r1ictin~ h which type of RNA has been produced.
Reference is now made to Fig. 5 which shows another
modification in the method outlined in Figs. 2 and 3 also int~n~le-l to
elimin~te production of co-~ tin~ short RNA transcripts having
sequence a' ~ ;l)ed from first DNA molecules. First DNA molecule 520
cc--l~ only a single-str~n~ç(l non-fim~tif-n~l promoter (P-). Assayed DNA
502 is added and allowed to hybridize with f~rst 520 and second 522 DNA
molecules and after addition of a ligæe, ligation product 526 is obtained.
To the reaction ll~ixLule a blocker molecule 536 is added c~ at its S'
end a sequence which is partially complemPnt~ry to part of the promoter
se~ nee (P~' partial)) linked to a sequence A' compl~m~nt~ry to sequence
A or to a part thereof. The blocker molecule 536 can hybridize only with
free first molecules 520 to yield hybrid S38 and cannot bind to hybrid 526
since in this hybrid se~ e A is already double-str~n~led Since P~' is only
partially complçm~nt~ry to the promoter, the presence of micm~tÇ~es in
hybrid 538 makes its promoter non-functi- n~l At ~is stage, DNA
molecules 540 co~-t~ a seqllence P~' complem~nt~ry to the full single-
str~n~l~d promoter of the first DNA molecules are added. Molecules 540 can
hybridize with hybrids 526 to give a hybrirli~tion product 542 having a
functional double-stranded promoter. How~ ., molecules 540 cannot
hybridize with blocked hybrid 538, since the promoter of the hybrid is
already partially double-str~nde~l By this modification free first molecules
are blocked from formin~ a functional double-stranded
SUBSTITUTE SH~ET (RULE 26~
WO 94129~1 :i.. PCT~US94/06034.
~43~
26
promoter so that when transcription re~nt~ 513 are added to the reaction
mixture, no short RNA transcripts are produced and only RNA triggering
molecules 510 are formed. The me~od outlined in this figure can be used
in combination with the method of Fig. 2 in which case blocker molecule
540 conlaills also sequence A" of Fig. 2 and is used to increase the
specificity of the method.
R~r~llce is now made to Fig. 6 showing a mo-lifir~tion of the
embodiment depicted in Fig. 1. In accord~lce with this embo-limrnt
~etection c;lls~lllble 604 co, ~-p~ ;~es a first DNA molecllle 620, a second DNAmolecule 622 and a third DNA molecule 623. These three molecules
comprise single-stranded sequences A, B, C which are complement~ry to
corresponding sequences A', B', C' in the assayed DNA sequence 602.
Following hybridization, a hyhri-~i7~tion product 624iS produced which is
formed from the first, second and third DNA molecules on the one hand and
the assayed DNA sequence on the o~er hand. Following an optional step
of li~ti~-n and a step of addition of transcription re~nt~ 613, a t1i~;~erin~
RNA molecule 610 is produced in a m~nn~r similar to that described in
Fig. 1.
RNA AMPLIFICATION ENSEMBLE
First embodiment of the invention
R~r~ ce is now made to Fig. 7 showing an RNA amplifica-
tion t;..~r~ hlc according to a first embo~im~nt of the invention. According
to this embo-lim~nt ~e tri~5~n~ RNA molecule 710 which is a product of
the detection en~ernble contains a'and b' sequences transcribed from DNA
se~ r~ A and B. Up~ - of sequence a' is sequence c'and downstream
of sequence b' is sequence y'. Sequence c' and y' which are ~anscribed
from sequences in the first and second DNA molecules, respectively, are
o~ seq~n~s compl~nPnt~ry to each other. Downstream of sequence
SUBST~7111 E SHEET (RULE 28)
WO 94/29481 216 4 3 2 a PCT/US94/06034 .
27
y' is sequence s. Sequence s is a sequence that serves as a strong stop
transcription sequence when molecule 710 is transcribed. Sequence s is
linked to a self-replic~tin~ X-RNA sequence. The complPmpnt~ry sequences
c'-~' bring to the formation of a loop form which functions to minimi~e the
il,hlr~ ce of seqllenres a' and b' to the seC~n~l~ry structure of the X-RNA,
which secon~l~ structure is necP,~ for its self-replicating activity. In the
presence of RNA ~ tion re~gPnt~ 713, triggering RNA molecules 716
are produced, co,l~ isillg an X-RNA sequence. The X-RNA sequence
serves also as the signal molecule, the presence of which is ~letecte(1 by
means known per se. Stop seql~Pnre s ~ ellls the sequences a'and b' from
being l~ c~;hed from m~lecllle 710. Owing to its self-replicating ~lo~clly,
large amounts of X-RNA mnlcc~ 716 are produced within a short period
of time. The presence of large (~ ;es of RNA will then serve as an
indication for the presence of the assayed DNA in the sample.
ce is now made to Fig. 8 which shows a modification
in the m~th~ of the first embo~ .l Accordil,g to this mo-lifi~tion, the
triggering RNA 810 comprises sequences a' and b' (tr~n~crihed from the
assayed DNA) linked to sequence s which serves as a stop signal to the
transcription as described above. Sequence s is linked to an X-RNA
seqll~n~e which can serve as a t~ 'e for the production of self-replicating
X-RNA. This se~ re is linked to a s~uellce capable of forming a hairpin
loop so that the hybridized arms of this loop form a fimctio~ promoter p+
in a manner similar to that previously reported for DNA (Kohli V. et a.,
Anal. Biochem., 208, 223-227, 1993). In the presence of a transcription
re~g~?n1~ 813, this RNA promoter enables the pro-lllction of self-replicating
X-RNA 816, the detection of which signifies presence of the assayed DNA
in the ori~in~l sample.
SUBSTIl~IJTE SHEET (RULE 26)
wo 94ng48~ 4 3 2 0 PCT/IJ594/06034
Fig. 9 shows another modification of the first embo.lim--nt
According to this modification triggering RNA molecule 910 comprises in
addition to the a', b', s and X-RNA sequences (which were described with
reference to Fig. 8) the template strand of a promoter sequence p~'.
Optionally, DNA molecule 941, which comprises a sequence P- comple-
mentary to the single-stranded RNA promoter, is added and allowed to
hybridize with molecule 910, to form DNA/RNA hybrid molecule 933
having a double-str~n~led RNA/DNA promoter and an RNA sequence which
serves as a template. In the plesellce of ~.,...~f .;l~l;f)n reagents 913, transcript
916 which is a self-replication X-RNA sequence, is pro~lllce~l
Reference is now made to Fig. 10 which shows a third
modification of the first embo-lim~nt Tri~ge~ing RNA 1010 co,.l~.ises
seqll~ n~ç5, a', b', s and X-RNA as ~lesc~ribed above. A DNA molecule 1041
is added which co...~ es a double-str~n~led functional DNA promoter of
which the non-t~ ldle strand is linked to a DNA sequence complem- nt~ry
to all or to part of the X-RNA sequence of molecule 1010. The last
nucleotide or several nucleotides of the template strand of molecule 1041is
optionally an RNA nucleotide. Molecules 1010 and 1041 are allowed to
hybridize, and following ligation of the last RNA or DNA nucleotide of
molecule 1041 and the first nucleotide of hybrid 1010, hybrid 1033 is
f~rm~l In the presence of !~A~ ;on re~nt~ 1013 self-replicating RNA
1016 is produced.
Second emLc~ ent of the invention
Rer~,wlce is now made to Fig. 11 showing an RNA amplifica-
tion ensemble accoldillg to the second embo~limpnt of the invention.
Accordillg to this embo~liment the tri~rin~ RNA molecule 1110 product
of the detection en~emhle, co~ ills sequences a' and b' linked to the
trigg~in~ sequence p~' which is a single-stranded sequence complem~nt~ry
StlBSTITUTE SHEET (RULE 26)
WO 94/29481 ~1 6 4 3 2 0. : - ~ , PCT/US94/06034 .
29
to an essential part of the single-stranded promoter P~ of a fourth DNA
molecule 1148. Fourth DNA molecule 1148 contains at the 3' end of its
template strand a single-st~n-le~l promoter sequence P~', linked to a double-
skanded signal DNA sequence SIG, which is in turn linked to a DNA
sequence capable of being transcribed to the tri~erin~ sequence p-' termed
'promoter-sequence" in the figure. In some cases P~ of molecule 1148 may
be partially double-str~n~le~l and is only single-str~n-1Pd in a part çe~çnti~l for
the promoter's function. When RNA molecule 1110 is added to molecule
1148, the tri~Pring seq~Pn~e p~' of m~lecnle 1110 hybridizes with sequence
P~ of molecule 1148 to form an RNA/DNA heteroduplex 1150 having a
double-stranded functional promoter P+ co~ .g of one DNA strand and
one RNA strand or partial RNA strand. Upon addition of transcription
reagents 1113, RNA transcript 1116, which is the signal RNA molecule
co~ l;sillg the RNA signal sequence "sig" and sequence p~', is prod~lce(l
RNA 1ranscript 1116 can in turn hybridize with the fourth DNA molecules
1148 to produce RNA/DNA hel~lo.lu~lexes 1151 which in the presence of
~e transcription re~nt~ 1113 causes prodlle~tion of more RNA Llallsc~
1116 in a self-amplifying m~nnPr. Thus, the amounts of signal RNA
molecules in the me~ lm increases rapidly and in a short period of time
large ~n~ntitiPs are produced. The signal molecule can then be detected by
means known per se, either by detectin~ of the presence of the specific
signal seqllPn~e or by merely d~1~ ...i..ing the ~lu~llily of RNA in a sample,
for example, by change in light absoll,~ce. The presence of the signal
RNA molecule indicates the exi.~tence of the assayed DNA in ~e ori~in~l
sample.
Third embodiment of the invention
R~r~nce is now being made to Fig. 12 which shows an RNA
amplific~tic n ~ le accordh~g to the third embo~liment of the invention.
SUB~TlTUTE SHEET (RULE 26)
WO 94/29481 2 ~ 6 ~S 3 2 ~ PCT/US94/06034
.
The RNA triggering molecule 1210, product of the detection en.cçmble,
comprises sequences a' and b' linked to sequences c'and d' which are
complem~ont~ry to the single-str~ntle-l sequences C and D in the fifth 1252
and sixth 1254 DNA molecules, ~*,e.ili~ely. Fifth DNA molecule 1252
comprises a double-stranded functional promoter P+ linked to a double-
str~n~ l sequence y linked to a single-stranded sequence C. Sequence y
and sequence C are complement~ry to each other. Sixth DNA molecule
1254 comrrices at the 3' end of the template strand a single-stranded
sequence D, linked to double-stranded sequences, C, D and a signal DNA
sequence.
The two DNA molecules 1252 and 1254 are allowed to
hybridize with RNA transcript 1210 to give an RNA/DNA heteroduplex
1256. In this hybri~li7~tion product molecules 1252 and 1254 are joined
together by RNA tr~n.ccrirt 1210. A ligase is added to ligate the ~ cçnt
ends of DNA molecules 1252 and 1254 to yield a ligation product 1258. In
the l,r~s~,lce of transcription re~ntc 1213, an RNA molecule 1216 is
produced. In this molecule sequence y' and c' which are complem~nt~ry,
form a loop. The signal se~ e sig in ms)lcc~lle 1216 can then be detecte~l
by means known per se. In addition, RNA molecule 1216 can be further
made to hybridize with more fif~ 1252 and six~ 1254 DNA molecules to
form an RNA/DNA heteroduplex 1260 optionally followed by ligation. In
the ~esellce of the transcription re~nt~ 1213 more RNA tr~n~c3 ipts 1216
are tr~n~cribe~l from hybrid 1260 which in turn cause form~tions of more
heteroduplexed 1260, and the reaction can continue in a self-amplifying
m~nn. r.
In the presence of a transcription system 1213, fifth DNA
molecules 1252, which compri~çs a fi-nrti~n~l promoter produces short RNA
~n~rirts 1262. These short RNA ~ however cannot illL~r~ with
the hyl~ li7~tic-n of ~e fifth DNA molecules 1252 with the tri~rin~ RNA
SUBSTITUTE SHEET (RULE 26)
WO 94/29481 , ; PCT/US94/06034
1210 or with RNA transcript 1216, wince due to the presence of comple-
mentary sequences c' and y' a loop is formed. -- i
Reference is now being made to Fig. 13 which shows a
modification of the third embotlimPnt of the invention. Fifth molecule 1352
compIises a functional double-stranded promoter P+ and a short single-
str~n~e(l 5' end of sequence M. The single-stranded sequence M may be a
5' end non-essçnti~l part of the promoter or a short linker sequence. Sixth
molecule 1354 is a DNA molecule co~ an X-DNA sequence coding
for a self-replicating X-RNA, which molecule is single-str~n~led in a small
region at the 3' end of the tç~nrl~te strand having a sequence N. According
to this modification the RNA transcript 1310, the product of the detection
ensemble, comprises the a'and b' sequences ~tt~rll~(l to a single-stranded
seq~ e m' which is compl~mlont~ry to the small single-str~n~le~l region M
in fifth molecule 1352, joined to a single-str~n~le~l seq~l~nce n' which is
compl~m~nt~ry to the single-stranded sequence N in sixth molecule 1354.
RNA transcript 1310 hybridizes with DNA molecules 1352 and 1354,and
option~lly with the aid of a ligase, joins the two DNA molecules to form an
RNA/DNA hchlo~l~lex 1364.
In the presence of Lla.ls~ tion re~gent~ 1313 heteroduplex
1364 is transcribed to two products: an X-RNA transcript 1366 which is
self-replicating in the presence of a transcription system 1313; and a
tr~n~c~pt 1368 having as its 5' end a sequence complem~nt~-y to 13 base
pairs of a promoter linked to an X-RNA sequence 1366 which is self-
replicating. RNA transcript 1368 can join additional fifth and sixth DNA
molecules to form RNA/DNA heteroduplex 1371 which heteroduplex in the
presence of a l.~ tion system can give rise again to X-RNA molecules
1366 and transcripts 1368. The detection is then pc.~ rl in a m~nner
similar to the above.
SV8Stlll~E SHEET (RULE 2B)
WO 94/29481 ~ 1 ~ 4 3 2 0 PCT/US94/06034
32
Fourth embodiment of the invention
The fourth embo-liment of the invention is shown in Figs. 14
and 15. The first, second and third emb~rlimPntc described above, make use
of the same detection ensemble and differ from one another in the amplifica-
tion ~neemhle. Against this, the fourth embo-1imçnt differs from the others
in both the detection as well as the amplification Pneçmbles.
The detection enePmhle in accordance with this embodiment
is shown in Fig. 14. First DNA molecule 1420, which in this embodiment
is c;~mpletely single-stranded, comprises at itS 3' end an &,I,illd,y
sequence C, linked to a short sequence of 1 to 5 bases termed ON and
linked to a seq~lPnre A. Second DNA molecule 1422 co.,~ Ps at its 3' end
a sequence B linked to an ~billal.y double-stranded sequence D-D'.
Sequence A of the f~rst molecule is complPmPnt~ry to
sequence A' in the 5' end portion of the assayed DNA and se ll~r~ .re B of the
second DNA molecule is co~ ..r to the seq~Pnre B' in the ~ g
3' end portion of the assayed DNA 1402.
At times, the sample c~ e also a seq~-enre 1402' c~ ;e;og
se.l~ .r~es A" and B" which are not fully comrl~ to se l~lr .reS A and
B in f~rst and second molecules 1420 and 1422, respectively. This may be
so, for example, in the case of genetic polymol~hisll,. The molecules in the
i~lu~ are allowed to hybri~li7e, pro~h~cin~ perfect hybridization products
1424 and iul~c.recl hybridization products 1424'.
In a similar manner as in the embo~im~?nt described in Fig. 3,
c-~nrlitione are provided so that e~senti~lly only imperfect hybrids 1424' are
melted. A blocker molecule 1425 is added to the ll~i~Lule which during
cooling hybri~li7es to free first DNA molecules 1420. The free first DNA
m~lec~ include bo~ first DNA molecules l)~S~llL a priori in the sample
and first DNA molecules which were freed from hybrid 1424' after melting.
Molecule 1425 comrri~es sequences ON' and A' complen ~nt~ry to
Sl)BSTlllJrE SHEET (RULE 26)
WO 94/29481 ~16 13 2 0 PCT/US94/06034
33
seqll~nre ON and A, respectively, in molecule 1420 and consequently hybrid
1427 is produced. In order to ensure that all free DNA molecules will be
blocked by blocker molecules 1525, an excess of the blocker molecules is
added.
To the sample are now added molecule 1429, 1431 and 1433
which together are able to form a functional promoter with the ligation
produet of hybrid 1424 which is 1426 while they are not able to form a
filnrtion~l promoter with hybrid 1427, thus avoiding the production of short
RNA transcripts having the sequence c-on-a.
Molecule 1429, termed herein '~romoter molecule", compri.~es
a double-s~nded promoter P+. One or a few of the RNA nucleotides at the
5' end of the template strand of the promoter are optionally RNA
mlrle~ti~es. This molecule can be produced by a nucleic acid syntll~si7~r.
The non-template strand of the promoter is linked to sequence E', C' and
ON'.
Molecule 1431 termed herein "ada~ter molecule " COI~p- ;~es
a single-stranded DNA sequence optionally having one or a few RNA
nucleotides at its 3' end. The purpose of the adapter molecule is to provide
a standard se ~ having an initial RNA nucleotide which can bind to the
RNA nucleotide of the promoter at its one end and to the first DNA
molecule (with the aid of a joiner molecule) on its other end, in a case
where an RNA molecule is required on the 3' end. When an RNA molecule
is not l~ Uil~;d, the adapter molecule provides a standard sequence common
to all reactions. ~lt~rn~tively, the sequence cont~in~d in the adapter
molecule may be added to each first DNA molecule when synthPsi7ed so
that the need for a separate molecule is elimin~ted
Mclecllle 1433 termed herein "joiner molecule", it co"l~l;ses
at its 5' end a part (e.g. a half) of E' and at its 3' end a part (e.g. a half)
of C'. This molecule serves to join adapter molecule 1431 and first DNA
SUBSTITUTE SHEET (RULE 2B)
WO 94/29481 ~ 3 2 0 PCT/US94/06034
34
molecule 1420. In addition, hybridization with this molecule renders the
.. . .
ON sequence essenti~l for hybridization of the promoter molecule 1429 to
first DNA molecule 1420. The fact that the ON sequence becomes ess~nti~l
avoids binding of promoter molecule 1429 to blocked hybrid 1427 in which
the ON sequence is covered, and thus the production of short c~nt~min~ting
sequences is avoided.
When the molecules are added to hybrid 1424, joiner molecule
1433 joins the 3' end of molecule 1420 (in the C sequence) and the 5' end
of adapter molecule 1431 (in ~e E sequence). After this joining, the
promoter molecule 1429 can hybridize to sequence E of the adapter
molecule 1431 and sequences C and ON in first molecule 1420. A ligase
then ligates the ~dj~c~nt ends of the RNA nucleotides both in promoter
molecules 1429 and adapter molecule 1131 (when present) resllltin~ in
hybrid 1435. ~dj~Gent RNA nucleotides can be ligated by the T4 DNA
which is known to be able to ligate bclweell RNA Nucleotides (Moore et
al., Science, 256~ 992-997, (1992). T,i~tion that occurs when the 3'
molecule is comprised of RNA and the S' molecule is comrri~es of DNA
and also efficiently ligated by T4 DNA ligate (Nath and Hurwitz, J. Biol.
Chem., 249, 3680-3688 (1974).
Promoter molecules 1429, together with the rem~inin~
co~ ullell~ cannot bind to blocked hybrid 1427 since in this hybrid the
sequence ON is blocked, which blockage pl~ ve.ll~ hybridization.
In the presence of transcription system 1413, RNA ~an-
script 1410 is produced.
~ he amplifi~ti~n ensemble is shown in Fig. lS. The ensemble
co~ lnises promoter mnlccllle 1529', being the same as molecule 1429 in
Fig. 14, and an opposite promoter molecule 1580 that is a promoter which
is a double-stranded promoter linked to a sequence D', complem~nt~ry to
sequence d' in ~e transcript 1510. Transcript 1510 being id~ntis;~l
SUBS17ME SHEET (RULE 26~
WO 94/29~1 ~ 16 ~ 3 2 0 PCTnJS94/06034
to 1410 in Fig. 14 hybritli7~s to D' sequence of opposite promoter 1580 and
with ~he aid of a ligase an RNA/DNA hybrid 1538 is formed. In the
presence of a transcription system 1513, a new RNA transcript 1539 is
tr~n~c~ed. The template of this transcript is the RNA sequence of hybrid
molecule 1538. It is known that RNA can serve as a transcript for RNA
pro~hlction (Leary S.L. et al., Gene, ~, 93-6 (1991). Transcript 1539 can
hybridize with promoter molecule 1529~ to give hybrid 1542. The product
of hybrid 1542is again molecule 1510 which can a~tiv~l~ opposite promoter
1580 and so on. In this embo~lim~nt the transcription product of each
hybrid 1538 or 1542 a~;liv~les the reciprocal promoter in a "ping pong"
m~nn~r.
The modification of the amplification en~mhle of the fourth
embodiment is now shown in Fig. 16, with all identical elements to those
of the ~ri~n~l ~mrlifir~tiQn en~Pn hle shown in Fig 15 having the same last
two digits.
RNA l~ sc~ 1610 product of the ~3etecti~-n ~n~emhle .,'
comprises in the 5' ~ 3' direction the following sequences: one variant of
the dis se ll~P ~ee - a dis2' se~ ce which is tr~n.~çrihe-l from one variant of
the DNA initiation sequence; e', c', on', a', b', d' which are the same as
e~l~ined in Figs. 14 and 15; and dis sequence which is another variant of
the dis sequence.
To the re~ction llliXI~ , iS added one type of rybozyme 1690
termed "r~boz~mel " which is able to sperifir~lly cut the dis1 sequence from
the 3' end of molecule 1610 to give h-~.rn~e~l RNA molecule 1611. RNA
molecule 1611 reacts with opposite promoter 1680, which comprises a
double-s~n-led pr(~motPr, the coding strand of which is s~ rh~1 to sequence
DIS1, and optionally with the aid of a ligase, gives hybrid 1638. In the
presence of transcription reagent 1613 RNA transcript 1639 is produced,
which may be ~letecte~l
SUBSTI~UrE SHEET (RULE 2B~
WO 94/29481 2! 1 6 a~ 3 2 a PCT/US94/06034
36
A second type of rybozyme 1591 termed "rybo~;yme2" which
is able to cut the dis2 sequence of transcript 1639 is used to give truncated
RNA molecule 1651.
RNA molecule 1651 reacts with promoter molecule 1629,
which contains a double-stranded promoter, the coding s~and of which is
attached to sequence DIS2, and optionally with the aid of a ligase gives
hybrid 1642. In the presence of transcription re~g~nt~ 1613 RNA transcript
1610 is produced again and the whole amplification cycle can restart.
SUBSTITUTE SHEET (RULE 2~)
