CA2213622A1 - Detection of biomolecules - Google Patents

Detection of biomolecules

Info

Publication number
CA2213622A1
CA2213622A1 CA 2213622 CA2213622A CA2213622A1 CA 2213622 A1 CA2213622 A1 CA 2213622A1 CA 2213622 CA2213622 CA 2213622 CA 2213622 A CA2213622 A CA 2213622A CA 2213622 A1 CA2213622 A1 CA 2213622A1
Authority
CA
Canada
Prior art keywords
ribozyme
initiation
nucleic acid
sequence
catalytically active
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA 2213622
Other languages
French (fr)
Inventor
Nathan Asher
Yaron Tikochinski
Guido Krupp
Jacob Grinberg
Adam Friedmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Intelligene Ltd
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from IL11279995A external-priority patent/IL112799A/en
Priority claimed from IL11577295A external-priority patent/IL115772A0/en
Application filed by Individual filed Critical Individual
Publication of CA2213622A1 publication Critical patent/CA2213622A1/en
Abandoned legal-status Critical Current

Links

Abstract

The present invention concerns a method for detecting the presence of a catalytically active ribozyme in a medium. The detection of the catalytically active ribozyme may be a goal by itself, or the ribozyme may serve as a reporter for the presence of other biomolecules in an assayed sample. The detection is carried out in a catalytic system wherein the presence of the active ribozyme serves to produce other active ribozyme in a positive-feedback amplificatory manner.

Description

-. CA 02213622 1997-08-22 W O 96127026 1~~ .'02380 DETECTION OF BIOMOLECULES

FIELD OF THE INVENTION
The present invention concerns a mcthocl and kit for thc detection of the presence of catalytically active ribozymes in a medium. The method and kit of the invention may be useful within the framework of a method 5 and kit for the detection of the presence of specific biomolecules in a test sample.

BACKGROUND OF THE INVENTION
Detection of the presence of specific biomolecules, such as DNA
10 or RNA sequences, proteins, anti~ens, antibodies, etc., in a sample is required for a variety of expcrimental, diagnostic and therapeutic purposes.
A multitudc of assays are available for detecting proteinaceous biomolecules such as gel electrophoretogram, HPLC, affinity chromatography, as well as other assays which are performcd by use of an appropriately labelled probe.
1~ While such assays are satisfactory where the proteinaceous biomolecule to be detectcd is presellt in sufficicntly large quantitics, they are at times not sensitive enough to allow dCtCCtiOII of minute quantities of biomolecules.

SUBS 111 UTE SHEET (RULE 26) W O 96/27026 PCTnUS96102380 DNA or RNA sequences can be detected by the use of a labelled probe. Where the DNA or RNA sequences to be detected present in only very small amounts, they have to be amplified by methods such as LCR
(ligase chain reaction), SSR (self-sustained sequence replication) or PCR
5 (polymerase chain-reaction).
Although amplification methods such as PCR have had an extremely high impact on basic research, they have been slow in making the transition to the clinical setting. The primary reason for this is that the requirement for automation combined with the clinical environment of the 10 samples, have yielded processes that are complex, slow and expensive. The need for protein enzymes with their high sensitivity to environmental factors necessitates a very controlled environment in which they are to operate.
Typically, a clinical sample contains many components that can interfere with the enzyme's ability to perform its catalytic activity. In addition, the 15 standard methods that are used for sample preparation to release the nucleic acids, such as Guanidine thiocyanate or Phenol extraction are unsuitable for protein bascd enzymatic activity and it is thcrcforc nccessary to rcmove the target nucleic acid from sample preparation.
Ribozymes are typically RNA molecules having enzyme-like 20 catalytic activities that are usually of cleavage, splicing or ligation of nucleic acid sequences. The known substrates for ribozymes are RNA molecules although there have been some indications that ribozymes may act on DNA
molecules and on proteins.
Natural ribozymes which participate in intracellular reaction work 25 in cis, catalyzing only a single turnover, and are usually self-modified during the reaction. However, ribozymes can be engineered to act in trans, in a truly catalytic manner, with a turnover greater than one and without being self-modified. Two distinct regions can be identified in a ribozyme:
the binding region which givcs the ribozyme its specificity through SUBSTITUTE SHEET (RULE 26) . CA 02213622 1997-08-22 W O 96n7026 PCTAUS96/02380 - 3 -hybridization to a specific nucleic acid sequence (and possibly also to specific proteins), and a catalytic region which gives the ribozyme the activity of cleavage, ligation or splicing. Each class of ribozymes cleaves a dirr~ t sequence of nucleotides using a ~ tinct mech~ m of action.
S Each class is further distinguished by the number of nucleotide bases that are essential for its catalytic activity and by the degree of the specificity ofthe ribozyme and the target sequence (Robert H. Simons, Annual Revie~
of Bioche~zistry, 61, pp. 641-671, (1992)).
It has recently been proposed to use ribozymes in order to treat 10 ~ e~ces or genetic disorders by cleaving a target RNA, such as viral RNA
or messengcr RNA transcribcd from gcncs that should bc turned off. This method is proposed as an alternative to blockage of the RNA transcript by the use of antisense sequences. Owing to the catalytic nature of the ribozyme, a single ribozyme molecule cleaves many molecules of target 15 RNA and therefore therapeutical activity is achieved in relatively lower concentrations than those required in an antisense treatment (WO 96/ 3569).
The use of ribozymes for diagnostic purposes has been only seldomly mentioned. WO 94/13833 describes a method for detecting nucleic acid molecules in a solution by tailoring a specific ribozyme 20 molecule having two regions, one complementary to the nucleic acid sequence to be detected, and the other complementary to a co-target molecule bearing a detectable label. The ribozyme is able to specifically and reversibly bind both a selected target nucleic acid sequence and to the labelled co-target. When both the target and the co-target are bound, the 25 ribozyme undergoes a conformational change which renders it active and able to cleave the label off the co-target, and the free label can then be detected. Upon cleavage of the co-target, the ribozyme is able to re-associate with an additional co-target, cleaving more label and producing more detectable signals.

SU B STITUTE SH EET (RU LE 26) W O 96127026 PCTnUS96/02380 _ 4 _ Although the inventors of WO 94/13833 termed their invention "amplification of signal" there is actually no amplification in the number of ribozymes produced, but rather the reaction is purely an enzymatic reaction, wherein the catalytic substance (in this case the ribozyme) cleaves 5 the substrate (in this case the co-target) and then disassociate and cleave another substrate. There is no true amplification of the number of active ribozymes involved in the reaction occurs.

GLOSSARY
Below is a glossary of terms which are used in the following description and claims. However, this glossary should not be considered separately and for full comprehension of the various terms and the meaning in which these terms havc in the context of the invention, the glossary should be read in conjwlctioll with thc remain~lcr of the disclosure herein.
Ribozyme -- a nucleic acid molecule which possesses an enzyme-like catalytic acivity. The term ~'ri~o7y~7te" as used in the art generally refers to RNA molecules having a catalytic activity although in the context of the present invention this term is used to denote a catalytically active (enzyme-20 like) oligonucleotide in gcneral. The ribozyme of the invention may thusbe an RNA molecule, may be an oligonucleotide comprising dNTPs or composed completely of dNTPs and may also comprise a variety of non-naturally occurring nucleotides such as IsoG or IsoC 5'-0-(1-thio-triphosphate) nucleosites and 5-0-methyl nucleotides. The ribozyme which 25 may be used in accordance with the present invention may be comprised exclusively of nucleic acids as described above or may require a co-factor for their catalytic activity. The ribozymes may have a catalytic activity of cleavage, ligation, splicing or splicing-out (removal) of oligonucleotide SUBSTITUTE SHEET (RULE 26) -W O 96n7026 r~l/U~96/02380 sequence, addition of groups to oligonucleotides, rearrangèment of nucleic acid sequences, etc.

Assayed biomolecule - a molecule the presence of which in the test sample S is to be detected. It can be an oligonucleotide or a member of a recognition pair such as receptor/ligand, antibody/antigen, lectin/glycoprotein, etc.

Initiation ribozyme - The ribozyme which initiates the reaction where more ribozymes arc produccd, evcntually Icacling to the gencration of a 10 detectable label. Where the method of the invention is used to detect biomolecules, the initiation ribozyme is part of the detection system (see below), and serves as a reporter for the presence of the assayed biomolecule, since only in the presence of said assayed biomolecules it is either generated or it becomes catalytically active. The prcsence of an active initiation 1~ ribozymc activates thc catalytic system (scc bclow).

Detection system - the combination of molecules and reagents that enable the production or activation of a catalytically active initiation ribozyme, which serves as a reporter for the presence of the assayed biomolecules in 20 the test sample. In other words, in the presence of the assayed biomolecules, following a reaction or a cascade of reactions, a catalytically active initiation ribozyme is eventually generated. The presence of the initiation ribozyme is verified in the catalytic system (see below) where it brings to the generation of more active ribozymes in an amplificatory 5 manner (the ribozymes themselves or a product of their catalytic activity, e.g. a free label is then detected at the final stage of the assay).

Inactive ribozyme - a potentially catalytically active ribozyme which cannot exert its catalytic activity (cleavage, splicing, ligation, etc.) until it SUBSTITUTE SHEET (RULE 26) W O 96/27026 ~ 6/02380 has been modified, or until the conditions in the medium have not been amended to such in which it becomes active.

Activation - rendering an inactive ribozyme catalytically active by some 5 kind of catalytic action (cleavage, splicing, ligation, addition of groups, rearrangement), or by change of external conditions (such as addition of magnesium ions).

Inhibitory moiety - a moiety which may at tim~s be present in the second 10 complex molecule (see below) and which when present renders the initiation ribozyme inactivc. The inhibitory effcct of the inhibitory moiety can be terminated by its modification or removal from the complex molecule.

15 Complex molecule - a molecule which forms part of the detection system in accordance with an embodiment of thc invention referred to herein as the "activation embodiment". In one mode of carrying out the activation embodiment, a '~irst comple~- molecule" is being used, which comprises an initiation ribozyme which is a priori, catalytically inactive (for example, due 20 to lack of magnesium ions in the medium) linked to a sequence capable of being cleaved by an active initiation ribozyme and comprising in addition a recognition biomolecule (see below). In another mode of carrying out the activation embodiment, a "second complex molecule" is being used, which comprises an initiation ribozyme which is a prioli, catalytically 25 inactive and is linked to a sequence capable of being cleaved by an active initiation ribozyme and comprising in addition a recognition biomolecule (see below). The'second complcx molccule further comprises an inhibitory moiety.

SUBSTITUTE SHEET (RULE 26) =

. CA 02213622 1997-08-22 W O 96127026 1~1~ 02380 Recognition biomolecule - a molecule capable of specifically recogni7~ing and binding to the assayed biomolecule. Where the assayed biomolecule is an oligonucleotide sequence, the recognition biomolecule is the complemen-tary sequence. Where the assayed biomolecule is a member of a recognition 5 pair (such as antigen-antibody) the recognition biomolecule is the other member of the pair.

First oligonucleotide - an oligonucleotide, typically a DNA molecule, which comprises from 3'-5': a double-stranded functional promotor, a 10 single-stranded sequence that codes for a sequence complementary to the sequence of the initiation ribozymes, a single-stranded sequence identical (with the necess~ry U-T replacements) with an RNA sequence capable of being cleaved by a catalytically active initiation ribozyme, and a single-stranded sequence complementary to the ~ '-part of the oligonucleotide 15 sequence to be detected.

Second oligonucleotide - an oligonucleotide, typically a DNA molecule, which comprises from 3'--~': a single-stranded 3'--part complementary to the oligonucleotide sequence to be detected, and a triggering oligonu-20 cleotide template (see below).

Triggering oligonucleotide template - an oligonucleotide sequence which is part of the second oligonuclcotide, thc transcription product of which is capable of hybridizing with the back promotor construct (see below).
Triggering oligonucleotide sequence - the transcriptional product of the triggering oligonucleotide template, capable of hybridizing with the back promotor construct (xee below) and after the back promotor has been SUBS 1~1 UTE SHLET tRULE 2~) W 096n7026 ~ 02380 completed, can bring to transcription of an oligonucleotide sequence to which it is attached.

Non-template strand oligonucleotide - the transcription product of the S oligonucleotide hybrid obtained by hybridization of the assayed nucleic acid sequence, the first oligonucleotide and the second oligonucleotide, and which comprises from 3'-5': a triggering oligonucleotide sequence, a sequence complementary to the assayed biomolecule, a sequence complementary to a sequence which can be cleaved by the initiation ribozyme, and a sequence 10 complementary to the initiation ribozyme.

Back promotor construct - a single-stranded promotor sequence attached to a single-stranded sequence capable of hybridizing with the triggering oligonucleotide sequencc. Aftcr hybridization with the triggering oligonu-lS cleotide sequence, and upon action of a suitable DNA polymerase, afunctional double-stranded promotor is created, which in the presence of a transcri~tion Systf~7~ (s;cc below) is capable of producing the final oligonucleotide transcriI)t (sec below).

20 Final oligonucleotide transcript - the transcriptional product of the oligonucleotide hybrids obtained following hybridization of the back promotor construct, non-template strand oligonuclcotide (after the promotor has been completed by a suitable DNA polymerase to a double-stranded functional promoter), and comprises from ~'-3': an initiation ribozyme 25 sequence, a sequence capable of being cleaved by said initiation ribozyme, a sequence that codes for thc complcment of the detected sequence on the assayed biomolecule, and a sequence that codes for the complement of the triggering oligonucleotide template. The initiation ribozyme in said final SUBSTITUTE SH EET (RULE 26~

W O 96/27026 ~-liU'r.'v23~C
_ 9 _ oligonucleotide transcript can cleave its adjacent sequence thus freeing itself and yielding a free, fully active initiation ribozyme.

Third oligonucleotide sequence - a nucleic acid sequence complementary S to the S'-part of the sequcnce to be dctectcd in thc assayed biomolccule.

Third composite molecule - a molecule used in the "assembly embodi-ment" of the invention, and which comprises the third oligonucleotide sequence linked to part of the initiation ribozyme. It optionally also 10 comprises a sequence cleavable by an active initiation ribozyme.

Fourth oligonucleotide sequence - an oligonucleotide sequence comple-mentary to the 3 ' -part of the sequence to be detected in the assayed biomolecule.
Fourth composite molecule - a molecule used in the "assembly embodi-ment" of the invention which comprises the fourth oligonucleotide sequence linked to part of the initiation ribozyme required to complete the part present in the third composite to obtain a complete catalytically active ribozyme.
20 It optionally also comprises a sequence cleavable by an active initiation ribozyme.

Catalytic system - An ensemble of molecules and reaction mixtures which in the presence of a catalytically active initiation ribozyme produces a - S detectable signal. This enscmble of moleculcs comprises a combination of composite molecules comprising a ribozyme which is a priori inactive. By one embodiment, the catalytic system comprises reagents and a combination of a first composite molecule and a second composite molecule (see below), comprising a first ancl SCCOlld ribozyme (inactive), respectively. The first SUBSTI I UTE SHEE~ (RULE 26) W 096/27026 ~ g6tO2380 -- 10-- .

and second ribozymes arc a priori inactive and either or both can be activated by a catalytically active initiation ribozyme. Active first ribozyme may activate inactive second ribozyme molecules, and active second ribozymes may activate inactive first ribozyme to cause amplification of the 5 number of active ribozymes in a positive feedback manner. Alternatively, the first and second ribozymes may be immobilized or spatially separated from each other and cleavage of one or both by the initiation ribozyme causes their release to the medium. Released, free ribozymes can free immobilized second ribozymes (for example by cleavage) and free second 10 ribozymes can in turn release and free first ribozyme, thus giving rise to self amplifying reaction cascade which rapidly yields an amplification in the number of active ribozymes in a positive feedback manner.
The catalytic systcm may also comprise in accordance with another emboidment, only one species of composite molecules which are 15 immobilized or spatially separated from each other in the reaction vessel or which are a priori inactive. An initiation ribozyme activates the inactive ribozyme or rcleases thc ribozymc from thc compositc molccule and active or released ribozymes then act to respectively activate inactive or free immobilized other ribozymes, then giving rise to a self amplifying reaction 20 cascade which rapidly yields an amplfication in the number of active ribozymes in a positivc fecdback manncr.
As a result of ribozyme activation or release, a detectable signal is produced which signal is indicative to the presencc of the initiation ribozyme in the medium.
First composite molecule - comprises a fiJst ribo~y~7le (see below), optionally labelled, linked to second nucleic acid se4uence (see below).

SU8 STlTUTE SH EET (RULE 26) . CA 02213622 1997-08-22 W 0 96r27026 ~~ 02380 Second composite molecule - eomprises a seco)ld ~ o~y~ (see below), optionally labelled, linked to afirst n~lcleic acid seq~lence (see below).

First nucleic acid sequence - an oligonueleotide sequenee whieh is part of 5 the seeond eomposite moleeule and whieh is a target for the eatalytie aetivity of the first ribozyme (see below). Following the eatalytic aetivity of the first ribozyme on the first nucleic acid sequenee, the seeoncl ribozyme (see below) is either released into the medium or becomes eatalytically active.
Second nucleic acid sequence - an oligonucleotide sequence which is part of the first composite molecule and which is a target for the catalytic activity of the second ribozymc (sec bclow). Followillg thc catalytic activity of the second ribozyme On the second nucleic acid sequence, the first 15 ribozyme (see below) is either relcase(l into the medium or becomes catalytically active.

First ribozyme - part of the first composite molecule -- is capable of cleavinL~ thc first IlUClCiC acid scqucllcc and is i~lcntical in its catalytic 20 activity to the initiation ribozyme. It is optionally labelled.

Second ribozyme - part of the second composite molecule - is capable of cleaving the seeond nucleie aeid sequenee and is optionally labelled.

25 Third ribozyme - a ribozyme whieh may form part of the catalytic system in accordanee with another embodiment thereof. The third ribozymes are initially inaetive. Initiation ribozyme acts to activate the third ribozyme by exerting itsi eatalytic activity thereon (in a manller to be explained below) SUBSmUTE SHEET (RULE 26) W O 96/27026 . ~11~_./02380 and the aetivated ribozymes ean then aet, to aetivate other third ribozymes in the eatalytie system.

Transcription system - ensemble of oligonueleotide, nueleotides, RNA
5 polymerase and reagents whieh, in the presenee of an oligonueleotide template bring to the transeription of an oligonueleotide transeript.

Fourth ribozyme - a ribozyme whieh is part of the eatalytic ~;ystem in aeeordanee with an embodiment thereof, and whieh onee catalytically aetive 10 ean ligate two parts of the fifth ribozyme (see below) to produee a eatalytieally aetive fifth ribozyme. The fourth ribozyme is eomposed of at least two eomponents, whieh are initially separated, and whieh are ligated together by Ihe fifth ribozyme (when eatalytieally aetive). Following sueh ligation, the fourth ribozyme beeomes eatalytieally aetive.
Fifth ribozyme - a ribozyme whieh is part of the eatalytie system eomprising the fourth ribozyme and which once catalytically active ean ligate together two parts of the fourth ribozyme to produee a catalytically aetive fourth ribozyme. The fifth ribozyme is composed of at least two 20 eomponents, which are initially separated and whieh are ligated together by the fourth ribozyme. Following such ligation, the fifth ribozyme becomes catalytically active.

Sixth ribozyme - a speeific example of the third ribozyme wherein the 5 inactive ribozyme carries an extra nucleic acid sequence, and is activated upon eleavage or splicillg-out (i.e. removal) of this sequence.

SUBSTlTUTE SH EET (RULE 26) W O 96127026 PCTnUS96102380 Seventh ribozyme - a ribozyme whcrcin the assayed llucleic acid sequence completes a mi~in~ portion essential for its catalytic activity, and thus once combined with the assayed sequence it becomes catalytically active.

The present invention provides a ribozyme-based signal-amplification method which is relatively simple to perform, is rapid and inexpensive. Unlike hitherto available detection-amplification methods, the method of the invention is also suitable for a point-of-care (POC) testing.
1() One a~va!lta~c of thc mcthod of the invention is in that ribozymes are active under conditions foulld in thc clinical environmcnt, e.L~. in bioligical fluids. Furthermore, as will be shown further below, the signal-amplification method in accordance with the invention does not require the observance of specific collcJitiolls in order to ensure specificity (strict observance of conditions is a must in prior art si~nal-amplification methods). Additionally, ribozymes are functional in various sample preparation cocktails, e.g. 1 M Guanadine thiocyanate as well as in a saturated phenol preparation, which usually inhibits function of other detection-amplification systems.
Ribozymes are composed of nucleic acid sequences and thus assay and probe sequences can be included in the ribozyme molecule. Further-more, it is possible to increase the specificity of the amplification process by engineering the ribozyme such that part of the assayed sequence itself is required for the ribozyme to exert its catalytic activity.
A very powerful technique, termed in the art as "in vitro evolution " has been successfully applied to ribozymes to produce a ribozyme with various catalytic activities and specificities. By such techniques, a vast array of potcntial ribozymc~ arc screcned for activity.
Those ribozymes that show activity are purificd for further rounds of SUBSTmJTE SHEET (RULE Z6) W 096/27026 1~l~ 02380 selection and after repeated rounds only the most potent candidates remain.
In traditional amplification techniques, following a choice of the enzyme, the environment where the enzyme is to operate, i.e. the sample-comprising medium, has to be modified to allow proper activity of the enzyme. In the case of ribozymes, using in vitro evolution, it is possible to select a ribozyme which is highly active in a dcsired clinical (biulogical) medium, by performing the in vct)-o evolution in a selcction medium which is identical in its composition to the clinical sample.
The present invention provides a sensitive method for the 10 detection of a cataytically active ribozyme (referred to herein as "initiation ribo7yme") in a medium. Detection of the presence of a catalytically active initiation ribozyme may be a goal by itself, although usually the catalytically active initiation ribozyme serves as reporter for the presence of other biomolecules in the tcst samplc. Once a mcdium comprising an active 15 initiation ribozyme is introduced into a catalytic system in accordance with the invention, there results a catalytic reaction cascade which gives rise to an exponential amplification in the number of active ribozymes. The catalytic system comprises ribozymes which are either inactive or spatially separated from one another such that they camlot exert their catalytic 20 activity; the initiation ribozymc frecs or activatcs ribozymes of the catalytic system which in turn free or activate respectively, further ribozymes of the system. The ribozymes either carry a detectable label or the catalytic activity causes generation of a detectable label, which then serves as an indication of the catalytic cascade which occurred in the system.
According to embodiments of the present invnetion in which the ribozymes are initially immobilized, the presence of free ribozymes in the reaction medium may serve as a dctectable signal by itself. In accordance with another embodiment, each active ribozyme is made to carry or produce a detectable label and thcse thcn serve as the dctcctable signal.

SUBSTmJTE SHEET (RULE 26) Accor~ling to the method of the invelltioll there is very little false positive signal, i.e. Iow noise Ievel; furthermore the method of the invention enables the detection of several biomolecules in a single assay system.
The present invention thus provides a method for detecting the 5 presence of a catalytically active initiation ribozyme in a medium, compris-ing the steps of:
(a) providing a catalytic system comprising (aa) ribozymes which are a pri~ri catalytically inactive or spatially confined such that they cannot exert their catalytic activity on their target; the target of the ribozymes being other ribozymes of the catalytic system, their catalytic activity on such other ribozymes causing either:
(i) activation of inactive ribozymes, (ii) release of spatially confined ribozymes to allow them to reach their targets;
at least some of the ribozymes of the catalytic system being a target of the catalytic activity of the initiation ribozyme, the catalytic activity of theinitiation ribozyme on said some of the ribozymes being that of(i) or (ii) above; ~nd comprising (ab) a detectable label having detectable properties such that the catalytic activity of the ribozymes causes a change in the detectable properties;

(b) contacting the medium with said catalytic system;
(c) providing conditions permitting said catalytically active initiation ribozyme and catalytically active ribozymes of the catalytic system to exert their catalytic activity, whereby the presence of a catalytically active initiation ribozyme gives rise to a reaction cascade in which ribozymes of the catalytic system are activated or freed into the medium; and SU BSTlllJTE SHEET(RULE 26) CA 022l3622 1997-08-22 W O 96/27026 l~l1~ 2380 - 16 -(d) detecting said detectable properties, a change in said properties being an indication of the presence of an active initiation ribozyme in said medlum.
The prime utility of the ribozyme detection method of the 5 invention, is within the framework of an assay designed to detect the presence of a biomolecule such as: a specific nucleic acid sequence, a member of a binding couple such as antibody-antigcn, sugar-lectin, etc., in a biological sample. Such an assay may conceptually be thought of as comprising two distinct components (although these components may be 10 included physically together in a single reaction vessel): a detection system and a catalytic system. In such an assay, the presence of the assayed biomolecule brings to the production, in a manner to be described further below, of a catalytically active initiation ribozyme in the medium. The catalytically active initiation ribozyme then acts as a reporter molecule in the15 catalytic system, giving rise, following a reaction cascade which amplifies the number of active ribozymcs, to the appearance of a detectable label in the reaction medium as generally described above. The appearance of such a detectable labcl in the mcdium of the catalytic system thus indicates the presence of the assayed biomolecule in the original assayed biological 0 sample.

DETAILED DESCRIP~ION OF THE INVENTION
In accordance with the invelltion, novel use is made of ribozymes.
In the most gcneralizcd scnsc, a catalytic system comprising ribozymes is 5 used for the detection of thc prescnce of a catalytically active initiation ribozyme in an assayed medium. Detection of the presence of a catalytically active initiation ribozyme in all assayed medium may be a goal by itself, for cxamplc, in thc proccss of prcparation of ribozymes by in vitro evolution.
In addition, in accordance with a prcfcrrcd embodiment of the invention, the SUBSTITUTE SH E~T (RULE 26) =

W O 96127026 PCTnUS96/02380 catalytically active initiation ribozyme serves as a reporter ~or the presence of an assayed biomolecule (other than the initiation ribozyme) in an assayed biological sample.
The ribozymes used in accordance with the invention may be S comprised entirely of RNA. At time it is possible also to replace some of the ribonucleotides ( "rNTPs ") in the RNA with deoxy nucleotides ("dNTPs") or some other naturally or non-naturally occurring nucleotides such as IsoG or IsoC 5'-0-(1-thiotriphosphate) nucleotsites and 5-0-methyl nucleotides. Such replacement is at times desired, for example, 10 to increase stability of the ribozyme to RNAase which are present in almost all biological samples. While this will not specifically be mentioned at each time, it is understood that thc term "ribo~yme" means to denote catalytic nucleotides composed entirely of rNTPs or catalytic oligonucleotides wherein some of the rNTPs have been replaced by dNTPs or other 15 nucleotides. The ribozyme may also be entirely composed of DNA (Breaker et al., Chenzishy and Bcology, 1(4):2~3-9, 1994).
The ribozymcs of the invention may comprise of nuclcic acid sequences as described above complexed with a non-nucleic acid molecule such as a protein, polypeptide, fatty-acid, dye, antibiotic, or a carbohydrate.
20 The non-nucleic acid moiety complexed with the ribozyme may serve as a co-factor for the ribozyme's catalytic activity.
In the following, usc of thc term "oligon~cleotide" will be made.
The oligonucleotides may, clepending on the context, be a DNA oligo-nucleotide (consisting entirely of dNTPs) or a RNA oligonucleotide 25 (consisting entirely of rNTPs). However, specifically in the case of RNA
oligonucleotides, it is at times desired to replace some or all of the rNTPs with dNTPs or other naturally and non-naturally occurring nucleotides.
The present invention provides, in its broadest sense, a method for the detection of the presence in a tested medium of a catalytically active SUBSTITUTE SHEET (RULE 26) W 096127026 ~1/~ 02380 initiation ribozyme. By "catalytically active", it is meant that a ribozyme is capable of carrying out a catalytic reaction such as cleavage, splicing, ligation, addition of specific groups such as phosphate to molecules, rearrangement of nucleic acid sequences and the like.
S The catalytic system in accordance with one embodiment for carrying out the invention comprises two species of ribozymes which are a priori inactive, but become catalytically active as a result of the exertion of a catalytic activity thereon. For example, each ribozyme may have a nick or break in a portion essential for its activity and prior ligation of this nickor break is thus required for its activation. The catalytic system in this case compriscs two spccies of ribozymcs, each spccics a pJ-i~l-i broken into two components and thus initially inactive. An active ribozyme of one species is capable of ligating the two components of thc second species of ribozyme thus rendering it active, and an active ribozyme of the second species of ribozyme is capable of ligating the two componcnts of the first species of ribozymes, thereby rendering it active. Then the activation proceeds by cross-ligation in a positive feedback amplificatory manner.
The first active ribozyme of one species may be produced by the initiation ribozyme which is a product of the ~Ictection system, in one of two routes. According to one routc, some of thc ribozymes of the first species are a priori fully assembled but cannot ligate the parts of the second species of ribozyme, since they are spatially separated therefrom, for example, as a result of being immobilizcd by means of a porous membrane, etc. The initiation ribozyme cleaves thc molecules of the immobilized fully assembled first species of ribozyme, and the free first species of ribozyme then ligates the second spccies of ribozymcs which Call in tum ligate members of the first species of the ribozyme which are not, a priori, assembled and so on.
According to thc sccond route, the initiation ribozyme is itself a ligating ribozymc which ligates from its parts, at least one species of the two SUBSTITUTE SHEET (RULE 26) W O 96/27026 ~ U~ 02380 ribozyme of the catalytic systcm, thus initiating the cross-ligation cascade.
In such a case there is no need to spatially separate various members of the catalytic system since until the initiation ribozyme is introduced to the reaction mixture no catalytic process can begin.
Another example are ribozymes which have a redundant sequence which renders the ribozyme inactive and it thus needs to be either cleaved or spliced-out for the ribozyme's activation. Further example are ribozymes which require sequence rearrangement or addition of specific groups for activation. Yet another examplc arc ribozymes which require reverse exon splicing, i.e. addition of a sequence internal to the ribozyme.
One species of ribozymes when in the active form, may activate inactive ribozymes of the SCCOlld species and vice velsa, as it possesses the catalytic properties (ligation, cleavage, splicing, rearrangement, etc.) required to moclify thc othcr spccics of ribozymc from an inaclivc to an activc form.
The two ribozymes may potentially possess the same type of catalytic activity (e.g. both are ligating rizoymes or both cleaving ribozymes, etc.) or may possess different types of catalytic activities.
Onc or both spccics of thc c~ pr~ inactivc ribozymcs is activated by a catalytically active initiation ribozyme. Prior to introduction of the initiation ribozyme to the medium, the catalytic system is çcsenti~lly silent as no catalytic activity takes place. In the presence of such an initiation ribozyme, a ribozyme amplification cascade begins since each active ribozyme generates in turn more active ribozymes in a positive feedback manner. Active ribozymes give rise to a signal which can be detected in a manner as described hereinbelow and such a signal is indicative to the presence of the original catalytically active initiation ribozyme in the medium.
The catalytic system in accordance with another embodiment for carrying out the invention compriscs two species of composite molecules, SUBSTITVTE SHEET (RULE 26) W O 96n7026 ~ 23~0 -- ')O --each comprising a ribozyme linked to a cleavable nucleic acid sequence.
The ribozyme in one species of composite molecules is capable of cleaving the nucleic acid sequence in the other species of composite molecules so that cross-cleavage between the two species of composite molecules is, in S principle, possible, while self-cleavage is avoided. However, prior to introduction of the initiation ribozyme to the medium, cross-cleavage does not result since the two specics of composite molecules are constructed so as to prevent mutual interaction between them, while cleaved ribozymes are able to interact with other composite molecules in the test vessel and further 10 release ribozymes to thc medium in a positive feedback manner.
Prevention of mutual interaction can be done, for example, by immobilizing each species of composite molecules to opposite sides of the test vessels; by linking each species of the composite molecule to different beads or different colloid particles having properties, e.g. size or other 1~ properties, e.g. the same electric charge (which repels the beads from one another) which prevents any kind of interaction between molecules attached to one with moleculcs attachcd to anothcr; by linking the composite molecules to moieties having the same electric charge, so that the electrical rejection between said moictics will prevent any interaction between the two ~0 composite molecules; by placing each species of composite molecules at opposite sides of a porous membrane which does not allow permeation therethrough of the full composite molecule, but allows free passage of cleaved ribozyme.
A catalytically active initiation ribozyme, either present in the test ''S medium a priori, or produced as a result of the presence of another biomolecule in a biological sample (such a ribozyme being in this case) ("a reporter- ribo~ynle") is ablc to clcave a spccific nucleic acid present in one or both species of composite molecules thus freeing the ribozymes of the catalytic system. The cleaved ribozymes are able to interact freely in the SUBSTlTUTE SH EET (RULE 26) . CA 02213622 1997-08-22 W O 96/27026 ~1l~','~02380 reaction vessel with the ribozymes from the other species of composite molecules, which in turn, can again cleave the ribozymes from the first species of composite molecules, thus creating a "ping-pong" cross-cleavage of ribozymes. Such cross-cleavage of ribozymes acts in a positive feedback manner, c~ in~ substantial amplification of the reaction. Either or both species of ribozymes typically bcars dctectablc labcls. Thc detcction of cleaved labels, indicates the presence of the catalytically active initiation ribozyme in the reaction mixture. Where the initiation ribozyme is a reporter ribozyme, detection of a frce label indicates thc presence of an 10 assayed biomolecule in the reaction mixture.
The catalytic system may comprise, in accordance with other embodiment, only OllC ~ipCCiCS of inactivc ribozymes or onc specics of composite moleculcs comprisill~ a ribozymc and a llUCICiC acid scqucncc cleaved by the ribozyme when converted into a free or active form. In an 15 analogous manner to that dcscribed above for the said one embodiment, each single molecule of the ribozyme is inactive until the catalytically active initiation ribozyme is introduce<l to the medium: for example, each inactive molecule is in the form of a closed-circle which may be opcned by cleavage or splicir,g-out of a strctch of nucleotides, by a catalytically active initiation 0 ribozyme. Activated (open) ribozymes thcn opcn and activate other such closed-circle moleculcs of the catalytic system.
In an analogous manner to that described above for the said another above second cmbodiment, each single species of the composite molecule may comprise a ribozyme positioned in an orientation which 25 prevents self-cleavage of the adjacent cleavable nucleic acid sequence, for example, by placing thc sequcnce immediately adjacent to the ribozyme.
The fact that there are no intcrvening sequences between the ribozyme and the cleavable scqucncc stcarically inhibits the cis clcavaL~e. (The cleavable sequence may also havc an invcrsed orientation, and cis cleavage will thus SUBSTITUTE SHEET (RULE 26) W O 96/27026 ~1~ 02380 not be possible). Howcvcr, rclcascd ribozymc may approach thc nucleic acid sequence in a correct orientation, and cleave it, thereby releasing more ribozymes to the medium. In order to prevent spontaneous trans cleavage of non-released ribozymes, it is possible to ensure spatial separation 5 similarly as indicatcd abovc.
Detection of the presence of activated ribozymes in the catalytic system may take various forms depending on the type of catalytic activity of the ribozyme. Where, for example, the activity is cleavage or spliced out, a label may be linked to the part to be cleaved or spliced out and detection 10 of such freed label is then an indication of the presence of the catalytically active initiation ribozymc in the mcdium.
In some cases the activation of the ribozyme brings to a change in the distance between two regions of the ribozyme, such as where two distant regions are brought together by ligation or rearrangement, by splicing 15 out of an interfering region, or wherein two initially adjacent regions are separated for example by opening of a closecl circle. In such case it is possible to attach a fluorescellt marker on one region of the ribozyme and a moiety, such as Rodamine which quenches the light emission from the fluroescent marker on the other region of the ribozyme. Rodamine has a O quenching effect on the light emission of a fluorescent label when the two are adjacent and no such effect when the two are separated. By monitoring the change in light emission of the fluorescent label, it is possible to determine whether the two regions are adjacent (for example in the case of a closed-circle inactive ribozyme) or separatecl (when the ribozyme has been 25 opened and activatecl).
The label may also be carried on a substrate which is not associated with the ribozyme and on which the catalytically active ribozyme may exert its catalytic activity. For cxample, the labcl may be carried on a nucleic acid sequence, which is cleaved or spliced-out as a result of a SUBSTITUTE SHEET (RULE 26) -W O 96~7026 ~liU'_-/02380 3 _ ribozyme's activity, whereby the label is released to the medium. The detection will in such a case be based on the presence of a free label in the medium.
The "ping-pong" cross activation, whether by cross-cleavage, S cross-ligation, cross-splicing, cross rearrangement or alternating cycles of various catalytic actions, substantially amplifies the reaction resulting in a signal that indicating in a short time whether the initiation catalytically active ribozyme was present in the medium. While prior art amplification-detection methods such as PCR or LCR require several hours to be 10 complete, the amplification-detcction method of the invcntion is completed in a much shorter time period.
Where the catalytically active initiation ribozyme serves as a reporter ribozyme for the presence of other biomolecules, a detection system is required in which a catalytically active initiatioll ribozyme is gencrated 15 only in the presence of the assayed biomolecule. This may be performed in one of the following embodiments, referred to herein as the ~ctivation embodiment", the "h-anscri~ti~n c~m~ 7zcnt the ''ase~7Zbly en~bodi--ment ' and the "completion ~mbodu7lent".
According to the activation embodiment, the initiation (reporter) ~0 ribozyme is ~ prio~i inactivc. This inactivity may bc a rcsult of thc absenceof magnesium ions, which are required for the ribzoyme's catalytic activity, from the medium; it may be a result of the presence of an inhibitory moiety in the medium; it may be a result of the presence in the medium of an oligonucleotide which hybridizes to sequence which is to be cleaved to 25 either activate or free the ribozyme into the system, which cleavage is not possible as long as thc scqucncc is doublc-strandcd; ctc. Accordillg to this embodiment, the ribozyme is linked to a recognition biomolecule which is capable of specifically recognizing and binding to the biomolecule which is to be assayed in the sample. For example, whcre the assayed biomoleucle SU8STITUTE SHEET (RULE 26) W O 96127026 ~ 96/02380 ' - 24 -is an oligonucleotide sequence, the recognition biomolecule is the comple-mentary sequence; where the assayed biomolecule is an enzyme, the recognition biomolecule may be a substrate; where the assayed biomolecule is an antigen, the recognition biomolecule may be an antibody which S specifically interacts with the antigen; etc.
The ribozyme linked to the recognition molecule is then allowed to interact with the assayed biomolecules, and unbound ribozymes are then separated and washed away. Such separation can be carried out, on the basis of size difference between the complex of bound ribozymes and 10 assayed biomolecules and that of free ribozymes; by, a priori, immobilizing the assayed biomolecules and then washing away free molecules of ribozymes; etc. After said separation, conditions are changed so as to activate the ribozyme, for example, by addition of lacking magnesium ions;
by modifying or removing thc inhibitory moicty to stop its inhibitory 15 activity; by melting the double-strandcd non-cleavable sequence to a single stranded cleavable sequence; etc. Only if the assayed biomolecule is present, ribozymes which are bound thereto are retained, and only these retained ribozymes are activated by an appropriate change of conditions.
The transcription embodiment of the invention can be utilized 20 where the assayed biomolecule is a nucleic acid sequence. The detection phase of this embodiment can be carried out generally as described in Israel Patent Applications Nos. ln~894 and 1118~7 (and their counterpart PCT
Applications Nos. WO 94/29481 and ) with the "triggering oligon~lcleiti~" being said initiation ribozyme. The detection system of 25 this embodiment comprises two oligonucleotide molecules, the first comprising a sequence complementary to the ~'- part of the assayed nucleic acid sequence and the second comprising a sequence complementary to the 3'- part of the assayed nuclcic acid sequence. The first oligonucleotide molecule comprises upstrcam from the sequence complementary to the SUBSTITUTE S~EET (RULE 26) . CA 02213622 1997-08-22 W O 96/27026 P~~ 5'~02380 assayed biomolecule, a functiollal promoter, a sequcnce that cocles for an initiation ribozyme sequence and a sequence that is capable of being cleaved by said detecting ribozymc (also esscntially a DNA sequence). The second oligonucleotide molecule comprises, downstream from the complementary S 3'- part of the assayed sequence, a triggering oligonucleotide template, the transcriptional product of which is capable of triggering transcription of sequences of initiation ribozymes as will be explained in detail hereinafter.
If the assayed biomolecule is not present in the test sample, then the triggering oligonucleotide sequence is not transcribed, since only presence of the assayed biomolecule brings togcther the two molecules required to produce thc appropriate template of said triggering oligonucleo-tide sequence: namely the first oligonucleotide molecule carrying the functional promotor and the second oligonucleotide molecule carrying the triggering oligonucleotide template. If the assayed biomolecules are present, and in the presence of transcription system, the triggering sequence is produced and in turn is able to bring about production of transcripts cont~ining initiation ribozyme linked to a sequence capable of being cleaved thereby. After self-clcavagc, these transcripts release to the medium catalytically active initiation ribozyme.
According to the assembly embodiment of the invention, also appropriate in cases where the assayed biomolccule is a nucleic acid sequence, the detection systcm compriscs a third oligonucleotide, comprising a sequence complementary to the ~'- portion of the assayed nucleic acid sequence, and a fourther oligonucleotide comprising a sequence complemen-tary to the remaining, 3'- portion of the assayed nucleic acid sequence.
Each of these oligonucleotidcs comprises also one portion (for example, half) of a ribozyme and both parts together constitute a full, functionally active ribozyme. In accordallce with this embodiment, the function of the assayed nucleic acid sequencc is to bring these two oligonucleotides SUBSTITUTE SHEET (RULE 26) =

W O 96/27026 ~ 96102380 . - ~6 -together, thus yielding a functionally active initiation ribozymc. Thus, in the presence of an assayed nucleic acid sequence in the sample, the functional initiation ribozyme will be generated which could then be detected in the catalytic system of the invention.
In accordance with the completion embodiment of the invention, the detection system comprises a seventh oli~onuclcotide and the assayed sequence, complexes with the seventh oligonucleotide to yield a catalytically active initiation ribozyme. For example, the assayed sequence may form part of the catalytic core of the ribozyme. Thus, in accordance with this embodiment, the ribozyme is a pr iori incomplete, and only in the presence of the assayed sequence it becomcs a complete, catalytically active ribozyme which may then be detectcd in the catalytic system.
The assayed sequcnce may complcte the ribozyme by hybridizing at its 3'-end to a sequencc on Onc sidc of thc ribozymc's missillg portion, and by hybridizing at its ~'-end to a sequence on the other side of the ribozyme's missing portion, thus bridging the missing portion and creating a functional initiation ribozymc.
The assaycd scquellcc may also bc able to complete the ribozyme's missing portion by ~ everse e~on splicing wherein the assayed sequence is inserted into the ribozyme through suitable cleavage and ligation reactions. Said reverse exon splicing may be carried out by other ribozymes present in the medium.
ln order to decrease the noise level of the method of the invention and decrease false positive results, it is possible to combine two 2~ or more embodiments of the invclltion to doubly ensure that no catalytically active initiation ribozymes are produced in the absence of assayed biomolecules. For examplc, it is possible to combine the assembly and activation embodiments of thc invcntion, whcrcby catalytically active ribozymes will be generatcd only as a result of two accumulative conditions:

SUBSTITUTE SHEET tRULE 26) . CA 02213622 1997-08-22 W O 96~7026 PCTrUS96/02380 - ~7 -assembly of a full ribozyme from its two parts in a magnesium-less mixture, and after washing away free incomplete ribozymes, activating of the full ribozyme by addition of magnesium ions.
The ribozymes used in most embodiments of the detection system S of the invention are in most cases universal, i.e. the same ribozyme can be used to detect different assayed biomolecules since the specificity is acquired by the attached recognition biomolecule (in the activation embodiment), or by the first and second oligonucleotide molecules (in the transcription embodiment) or by third and fourth oligonucleotide sequences (in the 10 assembly embodimcllt). Thc fifth oligolluclcotidc in thc case of the completion embodiment of the invention has to be tailor made for each specific nucleic acid to be assayed, since the sequence recognizing the assayed sequence is part of the ribozyme itself.
The present invention also providcs reagents required for carrying 15 out the above method as well as a kit comprising said reagents.
In the following thc invclltion will bc dcscribe~l with rcfcrcncc to ~;omc noll-limitill~, (3rawin~~,s and cxamplc~i:
In the drawings various symbols are used which in the context of the present invention have the following meanings:
Straight line ( ) - DNA strand Wavy line (~ RNA strand A,B,C, etc. ................ - sequences in the coding strand of a DNA
A',B',C', etc. ............. - sequences in the complementary non-coding DNA strand a,b,c, etc. ................ - RNA scqucnces a',b'c', etc. - RNA scqucnces complementàry to a,b,c S~JBSTITUTE SHEET (RULE 26) W 096/27026 ~ ,./02380 immobilization on a solid support * detectable label inhibitory moiety.

S BRIEF DESCRIPI'ION OF THE DRAWINGS
Fig. 1 shows an embodiment of the catalytic system of the invcntion comprising two species of composite molecules activated by cross-cleavage;
Fig. 2(a) and 2(b) show a catalytic system in accordance with an embodiment of the invcntioll comprisillg OllCSpCCiCS of composite molecule 10 activated by cross-cleavage or cross-splicing: wherein the ribozyme is in the form of a closed circle (Fig. 2(a)); wherein the ribozyme requires splicing for becoming active (Fig. (b));
Fig. 3 shows various manners in which composite molecules can be separated from one anothcr: by immobilization to distinct sites of the 15 reaction vessel (3A); by linkage to largc beads (3B); by linkage to charged moieties (3C); and by placing each species of composite molecules at opposite sides of a porous membrane;
Fig. 4 shows an example of the detcction system according to the activation embodiment of the invention wherein the ribozyme is activated 20 by addition of magnesium ions;
Fig. 5 shows another example of the detection system according to the activation embodiment of the invcntion wherein the ribozyme is activated by modification of an inhibitory moiety;
Fig. 6 shows a detection system in accordancc with the transcription 25 embodiment of the invention;
Fig. 7(a) and 7(1)) show a detection system in accordance with the assembly embodiment of the invelltion; where the rccognitioll sequence of the ribozyme hybridize to the assaycd nucleic acid sequence (Fig. 7(a)); or SUBSTITUTE SH EET (RULE 26) . CA 02213622 1997-08-22 W O 96/27026 ~~ -t02380 _ ~9 _ where the openecl stemp-ll of the ribozyme hybridizes with the assayed sequence (Fig. (b));
Fig. 8 shows an example of the catalytic system of the invention comprising two species of ribozymes activated by cross-ligation;
Fig. 9 shows yet another example of the detection system according to the activation embodiment of the invention wherein the ribozyme is activated by rendering the cleavable sequence single stranded.
Fig. 10 shows the cleavage results of a dcteclion system comprising the stem-II open ribozyme of Fig. 7(b);
Fig. 11 shows the cleavage results of a catalytic system comprising the closed-circle compositc molecule of Fig. (b); and Fig. 12 shows the cleavage results of ribozyme in the presence of untreated blood and denaturing agents.

DESCRIPI'ION OF SPECIFIC EMBODIMENTS
Catalytic system Refercncc is first ma~le to Fig. 1 showing one manncr of constructing the catalytic system of the invention. The catalytic system comprises two species of composite molecules 10 and 11. Composite 0 molecule 10 comprises one type of labelled ribozyme which will be denoted ribozyme A (12) linkcd to an RNA sequence denoted b (13). Composite molecule 11 comprises another type of labelle(J ribozyme which will be denoted ribozymc B (l 1) an~l an RNA sequence a (l5). Ribozyme B in molecule 11 is capablc of clcavinL~ sequellce b in molecule 10 and ribozyme A in molecule 10 is capable of cleavin~ sequence a in mole-cule 11. Initially molcculcs 10 ancJ ll are not able to interact since each is ~ immobilizcd to ~ distinct sitc of thc rcaction vessel. Initiation ribozyme 16 is also capablc of clcaving scqucnce b in molccule 10.

SUBSTITUTE SH EET (RULE 26) WO 96127026 ~,-lIU' _~ 102380 If initiation ribozyme is present in thc rcaction mixturc then sequence b is cleaved releasing free ribozyme A (12) to the reaction mixture. Free ribozyme A (12) is able to diffuse within the reaction vessel to cleave molecule 11, thus releasing to the reaction mixture free S ribozyme B (14). Free ribozyme B (14) is again able to migrate through the reaction vessel to cleave molecule 10 to release again free ribozyme A (12) and the cycle is repeated again and again in a positive-feedback m~nnP.r.
Since both ribozyme A and B are labelled, detection of either or both in the supernatant indicates thc presellce of initiation ribozyme 16 in the reaction 10 mixture.
The following is an example of molecule 10 comprising a ribozyme 12 of the type hammerhead linked to sequence 13 which is then labelled at 3'-end by biotin: (Capital letters: "-O-methylated; small letters: RNA) S'CCA cugauga gGCC GAAA GGCc gaa acGUguc CGU AAA-The following is an example of molecule 11 comprising another ribozyme l~ of the type hammerhead which is capable of cleaving O seqeuence 13 present in molccule 10. Ribozyme I 1 is linked to sequence 15 capable of being cleavecl by ribozyme 12 of molecule which is then labelled at its 3'-end by biotim (Capital letters: '-O-methylated; small letters: RNA) 5 5'-GAG ACG cugauga gGCC GAAA GGCc gaa acAC guc UGG AAA

Although ribozymes A and B are referred to as different ribozymes and sequences a and b are refcrred to as (:lifferent sequences, both the ribozymes and sequences may be actually idelltical. In such a case self-SUBSTITUTE SHEET(RULE 26) . CA 02213622 1997-08-22 W 096127026 l~ 3~02380 - 31 -cleavage in each molecule 10 or 11 is avoided by linking the ribozyme to its attached sequence in such a proximity which does not enable cleavage in cis, while free ribozymes are able to cleave composite molecules in trans.
This can be done by linking the ribozyme imme~liately adjacent to its S potentially cleavable sequence. This is because the ribozyme should be spaced from its potcntially clcavable scqucncc by xcvcral nuclctoidcs for efficient cleavage to take place. Therefore, when the ribozyme in the composite molecule is linked directly with no spacing to the cleavable sequence, there is no possibility of cis cleaving and the sequences can be cleaved only in trans while cnablillg only cleavage in trans.
Reference is now made to Fib. (a) which shows another alternative where only one species of composite molecules is present in the reaction vessel. The catalytic system comprises a single species of molecules 17', each comprising a ribozyme C' (18') and a cleavable sequence c' (19'). The molecule 17' is in the form of a closed circle and thus ribozyme 18' is initially inactive.
If catalytically active iniation ribozyme 16N is present in the media, it is able to cleave the s;cquence c' and open the ribozyme to become active. Open ribozyme 18' can in turn open, by cleavage, additional "O composite molecules 17' turning them active. Detection can be carried out by using a fluorescent label (F) and Rodamine (Rd). When the two are adjacent as in the closed molecule the light emission of the fluorescent label is quenched and when they are separated, as in the open molecule sequence, the light emission of the fluorescent label becomes stronger.
Reference is now made to Fig. (b) which shows yet another alternative for the catalytic system comprising only a single species of composite molecule. Ribozyme 17" has in its core region an extra nucleotide sequcnce 18" which renders the ribozyme inactive. At the SUBSTITUTE SH EET (RULE 26) W 096/27026 PCTrUS96/02380 ' - 3~ -tçrmin~l of the extra sequence is a blocking group 19 which does not allow spontaneous ligation of the open end.
Initiation ribozyme 16, which has the catalytic activity of splicing is capable of both cleaving out extra sequence 18 and blocking 5 group 19 and then of ligating the free ends to give a functional ribozyme.
Functional ribozyme is then capable of splicing other ribozymes in the reaction medium cau~inp an amplification of the reaction. Detection according to one option (Option 1) is carried out essentially as described in Fig. 2(a), but in this case, initially the Rodamine (Rd) at the fluorescent 10 group (F) are separated and only upon activation of the ribozyme they become adjacent, so that active ribozyme is detected by quenching of light emission. According to the second mode of detection (Option '~) the spliced out group comprising free extra sequence 18 and blocking group 19 carries a detectable label.
The advantages of the mode of Fig. (b) resides in a very low "noise" level since in order for an inactive ribozyme to become spontane--ously active (not in the presence of an initiation ribozyme) two spontaneous occurrences must happen spontaneous cleavage (at a probability of 10~6/min in 10 mM MgCI at physiological pH and at a temperature of 37~C) and ~0 spontaneous ligation (at a probability of 10 7/min) giving a very low probability for spontaneous activation (10 l3/min).
The label attached to either or both ribozymes A, B or C
(Fig. 1) may be any detectable label known in the art such as a radioisotope, a fluorescent label, an enzyme which in the presence of a substrate is '~5 capable of producing a color rcaction, etc.
Fig. 3 shows various manners in which the two composite molecules 10 and 11 of the first embodiment are positioned so as to avoid mutual interaction but allow interaction between free ribozymes 12 and 14 and composite molecules. It should be understood that the same principles SUBSTITUTE SH EET (RULE 26) W O 96127026 ~liU~96/02380 apply also to the other manner for constructing the catalytic system of the invention, i.e. where only one species of composite molecule is present, ,res~ te~l in Fig. 2.
In Fig. 3(A) molecules 10 and 11 are immobilized onto distinct and separate sites of the reaction vessel 18 while ribozymes 12 and 14 diffuse freely in the reaction mixture.
Fig. 3(B) shows molecules 10 and 11 which are immobilized onto beads 19 the size of which prevents interaction between said molecules.
However, free ribozyme 12 and 14 are able to diffuse freely in the reaction mixture and interact with the composite immobilized molecules.
Fig. 3(C) shows another example of separating composite molecules wherein composite molecules 10 and ll are attached to charged moieties bearing the samc charge 37. The electrical rejection between their attached moieties eliminates the possibility of intcraction between molecules 10 and 11. However, free ribozyme 12 and 14 which are essçn1i~1ly uncharged are able to interact with the composite molecule.
Fig. 3(D) shows yet another example of separating the composite molecules 10 and 11 by placing them at opposite ends of a porous membran~ 34 which ~crves as a sievc, blocking passage of large ''O molcculcs lO and ll whilc allowing passagc of thc ~mallcr frcc ribozymes 12 and 11.
Another manner for ensuring that the two composite molecules 10 and 11 cJo not interact is by the use of blocker molecules which are complementary to a specific sequence rendering it double stranded. According to this manner, composite molecule 10 comprises a blocker molecule which renders the cleavable sequence b and part of the catalytic region of ribozyme A double stranded. In the partially double stranded composite molccule lO, the ribozyme is not active due to the fact that its catalytic region is clouble strancled. Composite molecule 11 is SUBSTITUTE SHEET (RULE 26) W O 96/27026 ~-l/U~ 02380 blocked in a similar manner. If initiation ribozyme is present in the reaction mixture, it displaces a part of the blocker molecule present on the composite molecule 10, and then the initiation molecule is able to cleave sequence b.
Once sequence b is cleaved ribozyme A is also rendered active since the S partially displaced blocker molecule completely falls off composite molecule 10 turning its catalytic region to become single stranded and active. Active ribozyme A then displaces the blocker molecule of composite molecule 11 in a similar manner as described above cleaving the cleavable sequence a, turning Ribozyme B single stranded and active. Ribozyme B
10 then activates composite molecule 10 in a similar manner to the activation of the initiation ribozyme described above, and cross activation of the two composite molecules can then proceed.
Reference is now made to Fig. 8 which shows another alternative for constructing the catalytic system of the invention. The 15 catalytic system comprises two species of ribozymes 80 and 81 which are active when fully assembled but are inactive when separated to their parts 80a, 80b and 81a, 81b, respcctively. Full ribozyme 80 is capable of ligating ribozyme parts 81:~ and 81b to produce a full and active ribozyme 81. Full ribozyme 81 is capable of ligating ribozyme parts 80a and 80b to produce 20 a full and active ribozyme 80, so that cross-activation proceeds by cross-ligation.
Initiation ribozyme 86 or 86' is capable of creating a full and active ribozyme 80 cither by clcaving a full but immobilized ribozyme from a location wherein it is spatially separated from ribozyme parts 81a and 81b, 25 for example, in one of the mamlers specified in Fig. 3 (Fig. 8 top left) or by being ablc to ligate parts 80a and 80b to form the full and active ribozyme 80 (Fig. 8 top right).

SUBSTITUTE SH EET (RULE 26) . CA 02213622 1997-08-22 WO g6n7026 ~ 96/02380 DETECTION SYSTEM
Where the method of the invention is to be used to aid in the detection of biomolecules other than ribozymes, the invention includes also a detection system capablc of producing catalytically active initiation ribozyme only in the presence of the assayed molecule.
Fig. 4 shows one example of the activation embodiment of the invention. In this examplc, the assayc~J biomolcculc is an immobilizcd nucleic acid sequence A (41) for example a DNA sequence. Immobilization can be carried out in accordancc with any method known in the art, for example, with the aid of a cross-lillkill~ agcllt or by trapping the a~sayed nucleic acid molecule betwccn two porous membranes which permit passage of smaller molecules. Where the assayed biomolecule is a protein it can be immobilized onto bcads carryillg appropriatc trapping agents, such as suitable immobilized antibodies, directed against regions which are not required for detection, etc. Altematively, the assayed biomolecule can be immobilized onto a nitroccllulose shcct ancl another protein, such as albumin, should then be applied onto the nitrocellulose sheet in order to saturate all the shects' vacant locations and avoicl, in the next step, non-specific absorptioll.
The detection system also comprises first complex molecule 42 comprising ribozyme 43, linked to cleavable sequence c' (44) capable of being cleaved by active ribozymc, and further comprises sequence a' (45) complementary to the assayc~l sequence A (41). Ribozyme 43 does not self-cleave since molecule 42 is kept in magnesium-less reaction mixture which eliminates the ribozymes catalytic activity. This can be done, for example, by keeping complex molecule 42 in a magnesium-less EDTA--~ containing reaction mixture.

SUBSTITUTE SHEET (RULE ~6) W 096127026 ~l~ 02380 - 36 -An example of ribozyme ~13 linkcd to the cleavage sequence c' (44) of the type hammerhead (Capital letter~ 0-methylated; small letters: RNA; over- and undcrlined: DNA) 5 5'--GCAACAGTGGAGGAAAGCC UACguc UGG UACGU CCA cugauga gGCC GAAA GGCc gaa acGUAGU AAA

Molecules 41 and 42 are allowed to hybridize to give immobilized hybrid 46. Frec molcculcs 42 are washed away, and to the 10 immobilized hybrid 46 are added magnesium ions in a concentration sufficient to activate ribozymes. In the presence of such a concentration of magnesium ion ribozymc 13 is ablc to cleave scquence c', thus releasing itself to the reaction mixturc while leaving immobilizcd cleaved hybrid 47.
Free and catalytically activc ribozyme 43 can serve as the initiation 15 ribozyme in the catalytic system.
Fig. ~ shows another example of the activation embodiment of the invention. Assayed biomolecule 51, which comprises nucleic acid sequence A, for example a DNA sequence is immobilized as described above. The detection system comprises a second complex molecule 52 20 comprising ribozyme 53 linked to sequence c' (54) which can be cleaved by catalytically active ribozyme and sequence a' (55) complementary to sequence A in the assayed biomolecule. The complex molecule also comprises an inhibitory moiety 58 which, while present in its unmodified form, inhibits the catalytic activity of ribozyme 53. An example of an 5 inhibitory molecule is a nucleic acid sequencc complementary to part of the ribozyme. In the prcscllcc of such a scqucllcc, thc ribozymc folcls to an inactive three-dimensional form.
Molecules 51 and 52 are allowed to hybridize to give immobilized hybrid 56, and free molecules 52 are washed away. To 30 separate hybrid 56 are addcd modifying substanccs which are ablc to intcract SUBSTITUTE SH EET (RULE 26) W 096t27026 1~11~'_-'02380 with the inhibitory moiety 58 and modify it to an un-inhibiting form. For example, where the inhibitory moiety is a nucleic acid sequence which causes folding of the ribozyme, the modifying substance may be a sequence complementary to the inhibitory moiety, which hybridizes and blocks the S inhibitory moiety, thus allowing the ribozyme to rcfold to its active form.
Alternatively, the modifying substances may be substances able to remove or cleave the inhibitory moiety, thus termin~ting its inhibitory action.
Active ribozyme is then able to cleave sequence c, thus releasing itself from immobilized cleaved hybrid 57. Catalytically active free ribozyme 53 then serves as the initiation ribozyme in the catalytic system.
Reference is now made to Fig. 9 which shows another example of the activation embodiment of the invention. Molecule 91 comprises the sequence of the initiation ribozyme 90, attached to sequence c, cleavable by the ribozyme, and to sequence a and b which are capable of hybridizing with the assayed biomolecule bcing, for example the assayed DNA sequence of molecule 9~. Molecule 91 further comprises blocker DNA sequence 92 which comprises sequence B and C, capable of hybridizing to sequences b and c of molecule 91, respectively, to give a double-stranded structure.
Blocker DNA sequence 92 is linked via linker sequence 93. The ribozyme 90 is not capable of cleaving sequence c since its sequence region is double stranded (through hybridization to blocker sequence 92).
The assayed molccule 94 is then introduced to the reaction mixture. If the assayed biomolccule is complementary to a and b of molecule 91, then blocker sequence 92 is displaced by the assayed mole-~ 25 cule 94 to give hybrid molecule 95. In hybrid molecule 95, the cleavable sequence c is single stranded enabling the ribozyme to cleave it and thus to be freed to the reaction mixture as a catalytically active ribozyme 96 which serves as the initiation ribozyme in the catalytic system.

SUBSTITUTE SH EET (RULE 26) W 096/27026 1~-lIV~96/02380 ' - 38 -According to this embodiment conditiolls such as temperature, the length of the part of the recognition biomolecule b which is double stranded, etc. must be chosen with care so that the assayed molecule is able to displace blocker molecule 92 e~senti~lly only if the assayed biomolecule sequences A and B are perfectly matched to recognition sequences a and b.
Reference is now made to Fig. 6 which shows the transcription embodiment of the detection system of the invention appropriate where the biomolecule is a nucleic acid sequence. According to this specific embodiment a correct DNA template, which cventually brings to the 10 transcription of initiation ribozyme, is assemblcd from its parts only in the presence of the assayed nuclcic acid sequcnce. The detection system comprises a first oligonuclcotide molccule 61, being essentially DNA, comprising from 3' ,~'; a double-strallded promotor, a sequence R coding for the complementary sequcnce of the initiatioll ribozymc, a sequcnce C
15 coding for a sequence cleavablc by a catalytically active ribozyme, and a sequence Dl complementary to the ~ ' -part of the assayed nucleic acid scquence. The detection systcm furthcr comprises second oligonucleotide molcculc 62, bcing cssclltially DNA comprisillg from 3'~': a scqucncc D2 complementary to thc 3'-part of thc assayecl nuclcic acid scquence and a 20 triggering oligonuclcotide tcmplate (TRIG). if assayed nucleic acid se-quence 63 is present, ancl under appropriate hybridization conditions, sequence Dl of molecule 61 and sequence D2 of molecule 62 hybridize with sequences Dl' and D2', respectively, of the assayed nucleic acid sequence to give hybrid 6~.
In the presence of transcription system non-template strand oligonucleotide 65 is produced, comprising from 3'~': triggering oligonucleotide sequence tri~, dt' and dl' scquences, sequences c' r', complementary with those of thc clcavable nuclcic acid sequenccs and the initiation ribozyme, respectivcly.

SUBSTITUTE SHEET (RULE 26) W O 96n7026 ~liU~_-/02380 To the reaction mixture are added molecules of back promotor construct 66 comprising a single-stranded DNA promotor linked to a sequence capable of hybridizing with the oligonucleotide triggering sequence TRIG. Under appropriate conditions the back promotor construct 66 S hybridizes with molecule 65 to give hybrid 67. In the presence of a DNA
polymerase the single-stranded promotor is completed to give a functional double-stranded promotor in hybrid 68.
Hybrid 68 can serve, in the presence of transcription rcagents, as a template for the production of final oligonucleotide transcript 69 10 comprising from at its S'-end: an initiation ribozyme sequence r and a cleavable sequence c capable of being cleaved by said ribozyme.
The ribozyme r cleaves cleavable sequence c thus releasing itself to the surrounclillg mcdium in thc form of frcc ribozyme 70. Free ribozyme 70 can serve as the catalytically active initiation ribozyme in the 15 catalytic system.
One mode of thc asscmbly emboclimellt of the invention is shown in Fig. 7(a). Thc dctcctioll systcm comprises assayed biomolecule 71, comprisillg a nuclcic acid scqllellce A~ A~. In addition, the dctection systcm compri~ics a part of a ribozymc 72, comprisillg oligonu--O cleotide sequcnce a'~ complemcntary to sequellce Al ancl another part of a ribozyme 73 comprising oligonuclcoti~le sequence a2' complementary to sequence A2. The parts of ribozyme 72 and 73 together constitute a complete ribozyme if the two parts are asscmbled.
If assayed moleculc 71 is present in the medium, it can 25 hybridize to a, of ribozyme part - 72 and a2 of ribozyme - part 73 bringing the two parts togethcr to form functional ribozymc-assayed sequence hybrid 76, which may scrvc as an initiatioll ribozymc in a catalytic system, for example, by clcaving molcculc 77, which clcavage may be rcquircd to initiate the amplification cascade in thc catalytic system.

SUBSTITUTE SH EET (RULE 26) W 096r27026 . ~ /02380 Another mode of the assembly mode of the invcntion is shown in Fig. 7(b).
Ribozyme 79 of the hammer-head type has been constructed in which Stem-II has been shortened to have only one complementary nucleotide (represented by one line in the Fig. 2b) and the rem~inin~ portion of the stem have been opened to give arms a and b. Ribozyme 79 is capable of hybridizing with sequence 70 to form Stems I and III and then perform the catalytic activity, for example, cleavage of sequence 70.
However, ~ prioli ribozyme 79 is incapable of cleaving the cleavable 10 sequence 70 since its Stem-II is open and inactive. Arms a and b of the opened core have been constructed to be complementary to sequences A
and B of the assayed sequcnce, for example, DNA sequence 80.
In the presence of assayed DNA sequence 80, the arms a and b of the stem-II ribozyme 79 are hybridized to the assayed sequence to 15 produce a fully double-stranded Stem-II and thus the ribozyme becomes catalytically activc and can scrvc as an initiatioll ribozyme in a catalytic system where, for example, cleavable sequence 70 is part of an enzyme in the catalytic system which requires cleavage for its activation.

Example 1: Detection of ~n assayed nucleic acid sequence using a ribozyme with ~n open sten~
Ribozyme HH8 was dissected into two parts at the loop of 25 stemp-II. Each of the two ribozyme halves of HH8 (HH8-3 and HH8 5) has a different additional 17 bases tail sequence complementary to the LAMTAR0 DNA target molecule. In the presence of the target, the two halves are brought together and form an active ribozyme. In the LAMTAR0 target the two sequences complemelltary to the ribozyme halves are 30 continuous. In the other LAMTAR moleculcs (LAMTAR1 through SUBSTITUTE SHEET(RULE 26) . CA 022l3622 l997-08-22 W O 96/27026 r~l/~ 02380 . - 41 ~

LAMTAR4) the two sequences are separated by 1 to 4 non-complementary bases respectively. The ribozyme substrate is SB8-''4 which contains the sequence recognized by HH8.

(a) Method:
1. Sequences:
Oligodeoxyribonucleotides were synthesized on an Applied Biosystem 381A DNA synthesizer according to the manufacturer's recom-mended protocol. The Ampliscribe kit (Epicenter Tcchnologies) was used ln for all RNA synthesis 1~32 Pl UTP 13nnnCi/mmoll was purchased from Rotem Industries Ltd., Isracl.
DNA targets:
LAMTARO:
S'GCTCCGAGTCCACCTGCACGCCGACCAGTGCCGTÇTTCGGGA 3' LAMTARl:
S'GCTCCGAGTCCACCTGCACGCI'CGACCAGTGCCGTGTTCGGGA 3' LAMTAR~:
S'GCTCCGAGTCCACCTGCACGCI-l'CGACCAGTGCCGTGTTCGGGA 3' LAMTAR3:
~0 5'GCTCCGAGTCCACCTGCACGCl'ATCGACCAGTGCCGTGTTCGGGA 3' LAMTAR~:
5'GCTCCGAGTCCACCTGCACGCrA'l'ACGACCAGTGCCGTGTTCGGGA 3' UnderJined-complelllenlar~ to ril)oz~me half;
I~l)l(l-non-complemenlar)~ addilional sequence.
s RNA transcripts:
SB-''~ (substrale for HH8):
S'GGUCACAAUGUCGGUCGAGUUCCA 3' HH8-3 (ribozyme hal~:
5'GGCGACCCUGAUGAGGCCGCGUGCAGGUGGACUCG 3' HH8-5(ribozynle hal~:
5'GGAACACGGCACUGGUCGGGCCGAAACAUUAA 3' Underlined-complelllenlar~ to largel.
2. Preparation of RNA:
DNA oligonucleotides were syllthesized according to (1) above.
The oligonucleotides wcrc purified by electrophoresis O~ % polyacryl-amide 7M urea gcl, UV-shadowccl and clutcd ovcrnight at room tcmperature SUBSTITUTE SHEET (RULE 26) W O 96/27026 PCTrUS96102380 in O.SM Tris-CI (pH-7.0), ().l% SDS and ().l mM EDTA. Elutcd DNA
was precipitated with 0.1 volumes of 3M sodium acetate and 3 volumes of ethanol, resuspended in 1 mM Tris-CI (pH-7.0) and 0.1 mM EDTA and stored at -20~C until use. Purified oligonucleotides were annealed to 5 complementary non-template T7 RNA polymerase promoter oligonucleotide (TAA TAC GAC TCA CTA TAG G) in '~0 mM each by incubating at 95~C
for 15 seconds and at 7()"C, 6()oc, 55~C, 5()~C, 45~C, 40~C and 37~C for S min at each temperature. Transcription reaction mixture (S0 ml) contained 2 mg of annealed DNA, 1X reaction buffer, 10 mM Dithiothreitol, '' mM
ATP, CTP and GTP, 1 mM UTP, ~ mCi [a3~ P~ UTP and 1.1 mM MgCI2.
The mixture was incubated for 1 hr at 37~C and then for S minutes at 80~C
to deactivate the enzyme. The RNA was precipitated as described.
Transcripts were purified by elcctrophoresis on 15% polyacrylamide 7 M
urea gel. RNA was located by autoracliography and eluted as described.
15 Eluted RNA was prccipitatcd as dcscribed, resuspcnclecl in n.1X TE and counted in scintillation flour (Luma LSC).
3. Cleavage reaction:
Reactions ~10 ml) were normally carried out in the presence '~O of O.S pmol ribozyme, sn mM Tris-CI (ph-7.5), 1 mM EDTA (pH-7.5), 0.05% SDS and 30 mM MgCI2. The reactions were preincubated at 95~C
for 1 min in order to eliminatc alternative RNA conformations which may have formed during storage at -''()~C. The reactions were incubated at 37~C
for 1 hour and stopped by adding a dye solution containing 10 M urea and S 10 mM EDTA and put on ice. The samples were denatured at 80~C for 5 min and run on 15% polyacrylamide 7 M urea gel.

SUBSTITUTE SHEET(RU~E 26) W O 96/27026 PCTAUSg6/02380 ' - 43 -(b) Results:
The results of the polyacrylamide gel are shown in Fig. 10.
Ribozyme without a target did not produce appreciable cleavage. When the target was added, enhanced cleavage resulted. The enhancement was 5 greatest in LAMTAR4 Ribozymes tested.

Example 2 Catalytic system comprised a closed-circle composite molecule The SLS-prccursor ribozyme (Rz) is a circular Rz with 11 bp long stemp-II. The two recognition arms are connected in tandem with an extra cleavage base separating them. Therefore this robozime has no activity, but serves as a tcmplate for active ribozymes. Once it has been cleavecJ, the "open" ribozymc bccomcs activc (as shown schcmatically in 15 Fig. ~(b)). ln order to initiate the catalytic system an initiation ribozyme must be present. Spontaneous cleavage of RNA occurs at a rate of 1 event per 1 o6 molecules per minute in 30 mM MgCI2 at 37~C. A circular ribozyme spontaneously clcavcd at the cleavage sitc bccomcs active and is capable of serving as the tri~gcr.
o (a) Methods 1. Sequences:
Oligodeoxyribollucleotides were synthesized on an Applied Biosystem 381A DNA synthesizcr according to the manufacturer's recom-25 mended protocol. The Ampliscribe kit (Epicenter Tcchnologies) was used - for all RNA synthesis 1~32 Pl UTP [3000Ci/mmol~ was purchased from Rotem Industries Ltcl., Israel.
The RNA precursor (SLS's) sequellces were: 5' GGU CAG
CAG UCG AA ~Recognition arm I] X ~Recognition arm III] CUG AUG
30 AGA CUG CUG ACC A 3' SUBSTITUTE SHEET (RULE 26) W O 96/27026 PCTnUS96102380 ' - 44 -SLS code Recognition arm 1 Recognition arm III Cleavage Site 107 CGCG AAUU ~U
108 CUAG AAUU A~

215 UGCA ACGU ~U

2. Preparation of RNA
PrcparationofRNA was conductcd as describcd in Example 1 above.
3. Spontaneous cleavage reaction:
Reactions (1() ,1l1) were normally carried out in the presence of S pmol precursor ribozymc (SLS-transcripts), S0 mM Tris-CI (pH-7.5), 1 mM EDTA(pH-7.~),().n5~~SDSalld300 mM MgCI2. The reactions were 20 preincubated at 9~~C for 1 min in order to elimin~te altcrnative RNA
conformations which may havc formcd furin~ storage at -~noc. The reactions were incubatcd at 37~Cforl hour or ovcrnight and stopped by adding a dye solution containing 1û M urea and 10 mM EDTA and put on ice. The samples were denatured at 80~C for 5 min and run on 15%
''5 polyacrylamide 7 M urea ~cl.

(b) Results ' Thc rcsults of thc polyacrylamidc gcl arc shown in Fig. 11.
A panel of 8 different circular ribozymcs was examined for cascade activity.
The cascade was initiated, hy spolltaneous cleavagc as described above. As SUBSTITUTE SHEET (RULE 26) . CA 02213622 1997-08-22 W 096/Z7026 ~ 96/02380 - 4~ -shown in Fig. 1, one of the ribozymes (~313) demollstrated a functioning catalytic cascade resulting in amplification in a positive feedback manner.

Example III Ribozyme inhibition by blood and various materials S used for DNA preparation All amplification techniques require a sample preparation step to release nucleic acids and eliminate inhibition of the reactions by blood components. Several materials like SDS, phenol an~J guanidinium are used 10 in these preparations. An amplification re~ction without the need of a sample prep step is to be performed. Ribozyme activity in the presence of blood, with and without nucleic acid releasing agents, was tested. Both RNA-only and modified ribozymes were examined.

15 (a) Method 1. Oligonucleotides:
Oligodeoxyribonucleotides were purchased from the unit for molecular biology of the H<-clcl,lcs~ Hospital, Mount Scopus, Jerusalem. The modified ribozyme was synthcsized by RPI, Bouklcr, Co. The Ampliscribe 20 T7 transcription kit (Epicenter Technologies) was used for all RNA
synthesis. ~y32p~ ATP L6()(K)ci/mmol] an~l Lcx32P~ UTP L3()00Ci/mmol] were purchased from Rotem Industries Ltd, Israel. T4 Polynucleotide kinase was purchased from NEB, Beverly, Ma.

2. Ribozymes:
DS-~-RzA3-6: 5' GCAACAGTGGAGGAAAGCCUACgucUGGUACGUCCA
c~lga-lgagGCCGAAAGGCcgaaacGUAGUAAA 3' ., ribonucleoti~lcs;
~ U~ ,asc '2'-0-Mctll) ] modificalioll;
Undcrlincd u~",c,~as-;- dco~ ribonuc]colidcs.
HH8(RNA transcripl):
5'GGCGACCCUGAUGAGGCCGAAAGGCCGAAACAUUAA 3'.

SUBSTITUTE SHEET (RULE 26) W O 96/27026 PCTnUS96/02380 3. Labeling of modified ribozyme:
50 pmoles of the ribozyme, 10 units of T4 Polynucleotide kinase and '~0 ,L~Ci[y32P] ATP were incubated in lX reaction buffer in a total volume of 10 ,ul at '~5~C for 1 hr. The reaction was terminated by 5 incubation at 60~C for 10 minutes.

4. Preparation of RNA:
HH8 template DNA oligonucleotide was purified by electro-phoresis on 15% polyacrylamide 7 M urea gel, UV-shadowcd and eluted overnight at room temperature in 0.5 M Tris-CI (pH-7.5), 0.1% SDS and 0.1 mM EDTA. Eluted DNA was precipitated with 0.1 volume of 3 M
sodium acetate and 3 volumcs of ethanol, resuspenclcd in 0.1 XTE (1 mM
Tris-CI pH-7.0 and 0.1 mM EDTA) and stored at - 0~C until use. Purified oligonucleotide was annealed to complementary non-template T7 RNA
15 polymerase promoter oligonucleotide (TAATACGACTCACTATAGG) in '~0 ,uM each by incubatillg at 9~~C for 15 SCCOlldS all~l at 7n~C, 60~C, 55~C, 50~C, 45~C, 40~C and 37~C for ~ min at each temperature. Transcription reaction mixture (50 ,ul) contained '' ,uM of annealed DNA, lX reaction buffer, 10 mM Dithiothreitol, mM ATP, CTP and GTP, 1 mM UTP, 25 '~0 mCi [a32 P] UTP and 1.1 mM MgCI2. The mixture was incubated for 1 hr at 37~C and then for 5 minutes at 8()~C to deactivatc the enzyme. The RNA
was precipitated as described. Transcripts were purified by electrophoresis on 15% polyacrylamide 7 M urca gel. RNA was located by autoradiography and eluted as described. Eluted RNA was precipitated as described, 2~ resuspended in 0.1X TE and counted in scintillation flour (Luma LSC).

5. Cleavage reaction:
Reactions (1(),UI) wcrc normally carricd out in the presence of 0.5 pmol modified ribozymc or HH8, 50 mM Tris-CI (pH-7.5), 1 mD

SUBSTITUTE SH EET (RULE 26) . CA 02213622 1997-08-22 W 096/27026 1~1~ '02380 EDTA (pH-7.5), 0.05% SDS and 30 mM MgCl~ l of whole blood or O.~,ul of whole blood and the additional component were added (See Fig.
1). The reactions were incubated at 37~C for 1 hour and stopped by adding a dye solution cont~inin~ 10 M urea and 10 mM EDTA and put on ice. The 5 samples were denatured at 80~C for S min and run on 1~% polyacrylamide 7 M urea gel.

(b) Results The results are shown in Fig. 1'', wherein 0.5 pmol or 10 ribozymes were present in each sample an(i 1 ,ul blood was mixed with either 10% SDS 4 M guanidine isocyanate or phenol chloroform 1:1, 1 ,~cl of each trealed blood sample or the agent alone was added to the reaction test. The results of ribozyme activity is not inhibited by either 1% SDS, 0.35 M guanidine or by ~% phcnc)l~)chloroform whethcr blood is present or 15 not. Untreated blood dcgradcs thc RNA part of thc ribozymcs. These results show one of thc advalltagcs of the rihozyme based detection method of the invention in that denaturating agents used in the preparation of the catalytic sample do not hinder catalytic activity of the ribizoyme.

SUBSTITUTE SHEET(RULE Z6)

Claims (25)

CLAIMS:
1. A method for detecting the presence of a catalytically active initiation ribozyme in a medium, comprising the steps of:
(a) providing a catalytic system comprising (aa) ribozymes which are a priori catalytically inactive or spatially confined such that they cannot exert their catalytic activity on their target; the target of the ribozymes being other ribozymes of the catalytic system, their catalytic activity on such other ribozymes causing either:
(i) activation of inactive ribozymes, (ii) release of spatially confined ribozymes to allow them to reach their targets;
at least some of the ribozymes of the catalytic system being a target of the catalytic activity of the initiation ribozyme, the catalytic activity of the initiation ribozyme on said some of the ribozymes being that of (i) or (ii) above; and comprising (ab) a detectable label having detectable properties such that the catalytic activity of the ribozymes causes a change in the detectable properties;
(b) contacting the medium with said catalytic system;
(e) providing conditions permitting said catalytically active initiation ribozyme and catalytically active ribozymes of the catalytic system to exert their catalytic activity, whereby the presence of a catalytically active initiation ribozyme gives rise to a reaction cascade in which ribozymes of the catalytic system are activated or freed into the medium; and (d) detecting said detectable properties, a change in said properties being an indication of the presence of an active initiation ribozyme in said medium.
2. A method according to Claim 1, comprising the steps of:
(a) contacting the medium with a catalytic system comprising:
- a first and a second ribozyme, both being, a priori, catalytically inactive;
- said first ribozyme becoming catalytically active upon the catalytic activity of said second ribozyme and said second ribozyme becoming catalytically active upon catalytic activity of said first ribozyme;
- at least one of said first or said second ribozyme becoming catalytically active also upon catalytic activity of said initiation ribozyme;
- at least one of said first or said second ribozyme carries a label such that upon catalytic activity of the other ribozyme there is a change in the detectable properties of said label;
(b) providing conditions in which said initiation ribozyme and said first and said second ribozyme can exert their catalytic activity; and (c) detecting said detectable properties, a change in said properties being an indication of the presence of an active initiation ribozyme in said medium.
3. A method according to Claim 1, comprising:
(a) contacting the medium with a catalytic system comprising:
- a first composite nucleic acid molecule, comprising a first ribozyme of a kind capable of cleaving a first cleavable nucleic acid sequence, linked to a second cleavable nucleic acid sequence, cleavage of said second sequence releases said first ribozyme from said first composite molecule;
- a second composite nucleic acid molecule, comprising a second ribozyme of a kind capable of cleaving said second cleavable nucleic acid sequence, linked to a first cleavable nucleic acid sequence, cleavage of said first sequence releases said second ribozyme from said second composite molecule;
- at least one of said first or second cleavable sequences being cleavable also by said initiation ribozyme;
- said first composite and said second composite nucleic acid molecules being separated from one another to avoid contact between the two composite molecules;
- at least one of said first and said second composite molecules carries a detectable label, said label being released to the reaction medium following cleavage by the ribozyme comprised in the other composite molecule;
(b) providing conditions which enable a ribozyme cleavage and which allow migration of cleaved ribozymes between said first and said second composite nucleic acid molecules; and (c) detecting the presence of released labels, presence of released label in the medium indicating the presence of the catalytically active ribozyme in the medium.
4. A method according to Clain 1, comprising the step of:
(a) contacting the medium with a catalytic system comprising a third ribozyme being, a priori, catalytically inactive;
- each molecule of said a priori catalytically inactive third ribozyme becomes active upon catalytic activity of other molecules of catalytically active third ribozymes;
- said a priori catalytically inactive third ribozyme becoming active also by the catalytic activity of said initiation ribozyme;
- said third ribozyme carries a label such that upon catalytic activity of the other third ribozyme molecule thereon, there is a change in the detectable properties of said label;

(b) providing conditions suitable for said third ribozyme and said initiation ribozyme to exert their catalytic activity; and (e) detecting said detectable properties, a change in said properties being an indication of the presence of an active initiation ribozyme in said medium.
5. A method according to Claim 1, comprising:
(a) contacting the medium with a catalytic system comprising:
- a composite nucleic acid molecule, comprising a labelled ribozyme linked to a cleavable nucleic acid sequence in an orientation or at a location which prevents cleavage of said nucleic acid sequence by said ribozyme while present in the composite molecule, said nucleic acid sequence being eleavable by said ribozyme when it is present in a free form, thereby releasing said labelled ribozyme from said composite molecule;
- said cleavable nucleic acid sequence being cleavable also by said initiation ribozyme;
- said composite molecules being separated from one another to avoid contact therein between.
(b) providing conditions which enable ribozyme cleavage and which allow migration of cleaved ribozymes between said separated composite molecules; and (e) detecting the presence of released labels, presence of released label in the medium indicating the presence of the catalytically active ribozyme in the medium.
6. A method according to Claim 4, for detecting the presence in a medium of a catalytically active ribozyme having the catalytic activity of cleavage comprising:
(a) contacting the medium with a catalytic system comprising:

- a composite nucleic acid molecule comprising a ribozyme which has a cleavable nucleic acid sequence, said composite molecule being in the form of a closed circle and said cleavable nucleic acid sequence being cleavable by the composite molecule in an open form;
- said cleavable nucleic acid sequence being cleavable also by the initiation ribozyme;
- said composite molecule carrying a detectable label which changes its detectable properties upon opening of the closed composite molecule;
(b) providing conditions which enable ribozyme cleavage and migration; and (e) detecting for said detectable properties a change in said properties being an indication of the presence of catalytically active ribozyme in the medium.
7. A method according to Claim 4, for detecting the presence in a medium of a catalytically active initiation ribozyme having the catalytic activity of splicing comprising:
(a) contacting the medium with a catalytic system comprising:
- a sixth ribozyme having, in a region thereof essential for its catalytic activity, an extra nucleic acid sequence, said nucleic acid sequence renders the ribozyme inactive;
- splicing of said extra nucleic acid sequence renders the sixth ribozyme active;
- said extra nucleic acid sequence is capable of being spliced from the composite molecule by said sixth catalytically active ribozyme;
- said extra nucleic acid sequence is also capable of being spliced by catalytically active detectable label;

- said sixth ribozyme carrying a detectable label which changes its detectable properties upon splicing of the extra nucleic acid sequence.
(b) providing conditions which allow ribozyme splicing; and (c) detecting said detectable properties a change in said properties being an indication of the presence of catalytically active ribozyme in the medium.
8. A method according to Claim 3, wherein the first and second composite nucleic acid molecules are located on opposite sides of a porous membrane blocking their passage therethrough while enabling passage of first and second free ribozymes.
9. A method according to Claim 3, wherein the first and second composite nucleic acid molecules are immobilized on a substrate.
10. A method according to Claim 5, wherein each composite molecule is immobilized on a substrate.
11. A method according to Claim 9 or 10, wherein the substrate is a bead.
12. A method according to Claim 3, wherein the first and second composite nucleic acid molecules arc linked to moieties having the same electrical charges.
13. A method according to Claim 5, wherein each composite molecule is linked to a charged moiety, all moieties in the reaction mixture having the same charge.
14. A method of detecting the presence of an assayed biomolecules in a test sample comprising the steps of:
(a) contacting the sample with a detection system under conditions which enable production of catalytically active initiation ribozyme essentially only if the assayed biomolecules arc present in the sample; and (b) detecting the presence of the catalytically active initiation ribozyme according to the method of any one of Claims 1-5, the presence thereof indicating the presence of the assayed biomolecule in the sample.
15. A method according to Claim 14, comprising:
(a) providing a first complex molecule comprising: an initiation ribozyme under conditions wherein the ribozyme is catalytically inactive and a recognition biomolecule capable of specifically recognizing and binding said assayed biomolecule;
(b) contacting said first complex molecule with the test sample under conditions which allow binding between said recognition biomolecule and said assayed biomolecule while maintaining the conditions which render the ribozyme catalytically inactive;
(c) removing unbound first complex molecules;
(d) providing different conditions wherein the initiation ribozyme is rendered active; and (e) detecting the presence of the catalytically active initiation ribozyme according to the method of any one of Claims 1-5, the presence thereof indicating the presence of the assayed biomolecule in the sample.
16. A method according to Claim 15, comprising:
(a) providing a first complex molecule comprising: an initiation ribozyme, under conditions wherein the ribozyme is catalytically inactive, and a recognition biomolecule capable of specifically recognizing and binding said assayed biomolecule, said ribozyme being linked to a cleavable nucleic acid sequence capable of being cleaved by a catalytically activated initiation ribozyme, wherein cleavage of said cleavable sequence releasing catalytically active initiation ribozyme to the surrounding medium;
(b) contacting said first complex molecule with the test sample under conditions which allow binding between said recognition biomolecule and said assayed biomolecule while maintaining the conditions which render the ribozyme catalytically inactive;
(c) removing unbound first complex molecules;
(d) providing different conditions wherein the initiation ribozyme is rendered catalytically active to cause cleavage of said cleavable sequence and thus release the initiation ribozyme into the surrounding medium; and (e) detecting the presence of the catalytically active initiation ribozyme according to the method of any one of Claims 1-5, presence thereof, indicating the presence of the assayed biomolecule in the sample.
17. A method according to Claim 15, comprising:
(a) providing a hybrid molecule comprising: an initiation ribozyme linked to a cleavable nucleic acid sequence which is capable, when single stranded, of being cleaved by the initiation ribozyme, cleavage of said cleavable sequence releasing catalytically active initiation ribozyme to the surrounding medium, the hybrid molecule further comprising a recognition biomolecule capable of specifically recognizing and binding said assayed biomolecule, said cleavable sequence and a part of said recognition biomolecule being a priori, double stranded;
(b) contacting said hybrid molecule with the test sample under conditions which allow displacement of one strand of the double stranded part of the recognition biomolecule and the cleavable nucleic acid sequence by an essentially perfectly matched assayed biomolecule;
(c) providing conditions allowing for cleavage by catalytically active ribozymes to cause cleavage of single stranded cleavable sequence, thereby releasing catalytically active initiation ribozyme into the surrounding medium; and (d) detecting the presence of the catalytically active initiation ribozyme according to the method of any one of Claims 1-5, presence thereof, indicating the presence of the assayed biomolecule in the sample.
18. A method according to Claim 15, comprising:
(a) providing a second complex molecule comprising an initiation ribozyme and a recognition biomolecule capable of specifically recognizing and binding said assayed biomolecules, said ribozyme linked to a cleavable nucleic acid sequence capable of being cleaved by catalytically active initiation ribozyme, cleavage of said cleavable sequence releasing catalytically active initiation ribozyme to the surrounding medium, said second complex molecule also comprising an inhibitory moiety capable of inhibiting the catalytic activity of said initiation ribozyme;
(b) contacting said second complex molecule with the test sample under conditions which allow binding between said recognition molecule and said assayed biomolecule while maintaining the inhibitory moiety in its inhibiting form;
(c) removing unbound second complex molecules;
(d) modifying said inhibitory moiety in the bound second complex molecules so as to remove its inhibitory effect thereby causing cleavage of said cleavable sequence and releasing the initiation ribozyme into the surrounding medium; and (e) detecting the presence of the catalytically active initiation ribozyme according to the method of any one of Claims 1-5, presence thereof, indicating the presence of the assayed biomolecule in the sample.
19. A method according to Claim 15, wherein the conditions in (a) are essentially lack of magnesium ions and step (d) comprises adding to the reaction medium magnesium ions in a concentration sufficient to render said initiation ribozyme catalytically active.
20. A method according to Claim 14, wherein the assayed biomolecule is a nucleic acid sequence and the method comprising the steps of:
(a) contacting the sample with a detection system comprising:

- a first oligonucleotide molecule having a double-stranded promotor, a single-stranded oligonucleotide sequence being essentially DNA which is essentially identical with the initiation ribozyme sequence; a single-stranded sequence being essentially DNA which is essentially identical with a cleavable sequence capable of being cleaved by a catalytically active initiation ribozyme and a single-stranded sequence which is complementary to the 5'-part of the assayed nucleic acid sequence;
- a second oligonucleotide molecule having a single-stranded sequence being complementary to the 3'-part of the assayed nucleic acid sequence and further comprises a single-stranded triggering oligonucleotide template which can be transcribed to give a triggering oligonucleotide sequence, said triggering oligonucleotide sequence capable, in the presence of a back promoter construct and DNA polymerase, to trigger a reaction in a transcription system wherein the sequence to which it is attached is transcribed;
(b) providing conditions which allow the hybridization of said first and said second oligonucleotide molecule to the assayed nucleic acid sequence;
(c) adding a transcription system, under conditions which allow transcription, whereby triggering oligonucleotide sequence is transcribed, said triggering oligonucleotide sequence, in the presence of a back promoter, DNA polymerase and transcription system and under conditions allowing hybridization, DNA polymerization and transcription, brings to transcription of a final oligonucleotide transcript comprising initiation ribozyme linked to a cleavable sequence capable of being cleaved by catalytically active initiation ribozyme;

(d) providing conditions which allow cleavage of said cleavable sequence by said catalytically active ribozyme, to cause cleavage of said cleavable sequence and thus release itself into the surrounding medium; and (e) detecting the presence of the catalytically active initiation ribozyme according to the method of any one of Claims 1-5, presence thereof, indicating the presence of the assayed biomolecule in the sample.
21. A method according to Claim 14, wherein the assayed biomolecule is a nucleic acid sequence and the method comprising the steps of:
(a) incubating the test sample with a detection system comprising:
- a third composite molecule comprising a third single-stranded oligonucleotide sequence being complementary to the 5'-part of the assayed nucleic acid sequence, said third oligonucleotide sequence being linked to a part of the initiation ribozyme;
- a fourth composite molecule comprising a fourth single-stranded oligonucleotide sequence being complementary to the remaining 3'-part of the assayed nucleic acid sequence, said fourth oligonucleotide sequence being linked to a part of the initiation ribozyme required to complete the ribozyme linked to the third oligonucleotide sequence to give a complete initiation ribozyme;
(b) providing conditions allowing hybridization of said third and fourth oligonucleotide sequences with the assayed nucleic acid sequence and which enable assembly of a full initiation ribozyme from its parts; and (c) detecting the presence of the catalytically active initiation ribozyme according to the method of any one of Claims 1-5, presence thereof, indicating the presence of the assayed biomolecule in the sample.
22. A method according to Claim 14, wherein the assayed biomolecule is a nucleic acid sequence and the method comprises the steps of:
(a) incubating the test sample with a detection system comprising:
- a ribozyme of the hammerhead type wherein some of the double-stranded portion of Stem-II has been shortened and wherein the remaining double-stranded Stem-II is attached to two single-stranded sequences one being complementary to the 3'-end of the assayed nucleic acid sequence and the othe being complementary to the 5'-end of the nucleic acid sequence;
- said ribozyme being a priori inactive;
- hybridization of said two single-stranded sequences to complementary sequences renders the ribozyme catalytically active;
(b) providng conditions allowing hybridization of said ribozyme with said assayed nucleic acid sequence; and (c) detecting the presence of the catalytically active initiation ribozyme according to the method of any one of Claims 1-5, presence thereof, indicating the presence of the assayed biomolecule in the sample.
23. A method according to Claim 1, for detecting the presence in the medium of a catalytically active initiation ribozyme having the activity of cleavage comprising:
(a) contacting the medium with a catalytic system comprising:
- a fourth ribozyme of a kind capable of ligating parts of a fifth ribozyme, linked to a nucleic acid sequence cleavable by the initiation ribozyme, cleavage of said nucleic acid sequence releasing catalytically active fourth ribozyme to the surrounding medium;

- two parts of a fifth ribozyme which are capable of being ligated by the fourth ribozyme to give a catalytically active fifth ribozyme, said fifth ribozyme being of a kind capable of ligating parts of a fourth ribozyme;
- two parts of a fourth ribozyme which are capable of being ligated by the fifth ribozyme to give a catalytically active fourth ribozyme;
- said fourth molecule being a priori separate from the two parts of the fifth ribozyme to avoid contact thereinbetween;
- at least one of said fourth or said fifth ribozyme carries labels which changes its detectable properties upon ligation;
(b) providing conditions which enable ribozyme cleavage and which allow migration of the cleaved full fourth molecule to the two parts of the fifth ribozyme;
(e) providing or maintaining conditions allowing ribozyme ligation; and (d) detecting said detectable properties, a change in said properties being an indication of the presence of said initiation ribozyme in said medium.
24. A method according to Claim 1, for detecting the presence in the medium of catalytically active initiation ribozyme having the activity of ligation comprising:
(a) contacting the medium with a catalytic system comprising:
- two parts of a fifth ribozyme which are capable of being ligated by a fourth ribozyme to produce a catalytically active fifth ribozyme, said fifth ribozyme being of a kind capable of ligating parts of a fourth ribozyme;
- two parts of a fourth ribozyme which are capable of being ligated by a fifth ribozyme to produce a catalytically active fourth ribozyme; said fourth ribozyme being of a kind capable of ligating parts of the fifth ribozyme;
- either said two parts of the fifth ribozyme or said two parts of the fourth ribozyme are capable of being ligated by a catalytically active initiation ribozyme;
- at least one of said fourth or said fifth ribozyme carries labels which changes its detectable properties upon ligation;
(b) providing conditions allowing ribozyme ligation;
(c) detecting said detectable properties, a change in said properties being an indication of the presence of said initiation ribozyme in said medium.
25. A kit for use in the method of any one of Claims 1 to 24.
CA 2213622 1995-02-27 1996-02-27 Detection of biomolecules Abandoned CA2213622A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
IL11279995A IL112799A (en) 1995-02-27 1995-02-27 Detection of biomolecules
IL112799 1995-02-27
IL11577295A IL115772A0 (en) 1995-10-26 1995-10-26 Detection of biomolecules
IL115772 1995-10-26

Publications (1)

Publication Number Publication Date
CA2213622A1 true CA2213622A1 (en) 1996-09-06

Family

ID=26322997

Family Applications (1)

Application Number Title Priority Date Filing Date
CA 2213622 Abandoned CA2213622A1 (en) 1995-02-27 1996-02-27 Detection of biomolecules

Country Status (5)

Country Link
CN (1) CN1183812A (en)
BR (1) BR9607267A (en)
CA (1) CA2213622A1 (en)
NO (1) NO973926L (en)
RU (1) RU2139352C1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2782325B1 (en) * 1998-08-12 2002-05-24 Proteus METHOD OF IDENTIFYING POLYNUCLEOTIDE SEQUENCES AND / OR CORRESPONDING PROTEINS FROM A SAMPLE OF NUCLEIC ACIDS
CN109609505A (en) * 2019-01-14 2019-04-12 中国科学院成都生物研究所 A kind of hammerhead ribozyme of the shearing RNA screened in vivo

Also Published As

Publication number Publication date
NO973926D0 (en) 1997-08-26
BR9607267A (en) 1998-12-15
CN1183812A (en) 1998-06-03
RU2139352C1 (en) 1999-10-10
NO973926L (en) 1997-10-08

Similar Documents

Publication Publication Date Title
US5589332A (en) Ribozyme amplified diagnostics
JP2806455B2 (en) Nucleic acid probes containing improved molecular switches and assays and kits incorporating them
US6429301B1 (en) Use of a ribozyme to join nucleic acids and peptides
Ekland et al. The secondary structure and sequence optimization of an RNA ligase ribozyme
US5637459A (en) Systematic evolution of ligands by exponential enrichment: chimeric selex
AU2005322131B2 (en) Ligation-based RNA amplification
US6143503A (en) Use of a ribozyme to join nucleic acids and peptides
AU697317B2 (en) Detection of biomolecules
US6387617B1 (en) Catalytic nucleic acid and methods of use
US6207388B1 (en) Compositions, methods, kits and apparatus for determining the presence or absence of target molecules
JPH10510165A (en) DNA enzyme molecule
WO1996027026A9 (en) Detection of biomolecules
JP2002514080A (en) Enzyme DNA molecule
EP0177497A1 (en) Immobilization of nucleic acids
EP2137321B1 (en) Methods for detecting a target nucleotide sequence in a sample utilising a nuclease-aptamer complex
MXPA97006501A (en) Detection of biomolecu
US6214546B1 (en) Detection of biomolecules
CA2213622A1 (en) Detection of biomolecules
US20060141452A1 (en) Method For Synthesizing Single-Stranded Nucleic Acid
WO1993005184A1 (en) Dna/rna target and signal amplification
Li et al. In vitro selection of kinase and ligase deoxyribozymes
EP0710292A1 (en) Detection of nucleic acid sequences
Bashkin DNA enzymes: New-found chemical reactivity
Maillard et al. Isolation of New Ribozymes from Sequences
Collman Isolation of New Ribozymes from a Large Pool of Random Sequences

Legal Events

Date Code Title Description
FZDE Dead