EP1064405A1 - Composition contenant un adn codant pour un ribozyme et un substrat oligonucleotidique, et technique permettant de mesurer la vitesse de transcription - Google Patents

Composition contenant un adn codant pour un ribozyme et un substrat oligonucleotidique, et technique permettant de mesurer la vitesse de transcription

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EP1064405A1
EP1064405A1 EP99915650A EP99915650A EP1064405A1 EP 1064405 A1 EP1064405 A1 EP 1064405A1 EP 99915650 A EP99915650 A EP 99915650A EP 99915650 A EP99915650 A EP 99915650A EP 1064405 A1 EP1064405 A1 EP 1064405A1
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transcription
ribozyme
substrate
fluorescence
oligonucleotide substrate
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Andreas Jenne
Michael Famulok
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead

Definitions

  • compositions which contain a DNA sequence encoding a ribozyme, preferably a hammerhead ribozyme, and an oligonucleotide substrate which is cleaved by the ribozyme transcribed by the DNA sequence.
  • a FRET oligonucleotide is used, i.e.
  • an oligonucleotide substrate which is labeled with a fluorophoric group (reporter group) and a fluorescence-quenching group (quencher group), wherein after cleavage with the ribozyme, the quenching of the fluorescence of the fluorophore by the fluorescence-quenching group is prevented, i.e. a fluorescence signal is generated.
  • the present invention further relates to methods for measuring transcription rates, for example for determining inhibitors of transcription or transcription activators, using the composition according to the invention, and methods for measuring the catalytic activity of ribozymes.
  • the mechanisms of eukaryotic and prokaryotic transcription are usually investigated using methods in which the mRNA is synthesized in vitro, ie using appropriately prepared cell extracts, ("in vitro transcription").
  • Specially developed transcription vectors are used for the production of transcripts, which in addition to the reporter gene also contain the promoter for a corresponding RNA Wear polymerase.
  • the mRNA of the coding reporter gene must be detectable and quantifiable using suitable methods.
  • radioactively labeled nucleoside triphosphates are added to the cell extract, which are incorporated into the resulting mRNA.
  • the radioactively labeled mRNA is then isolated from the cell extract, electrophoretically separated on a polyacrylamide gel and visualized and quantified by autoradiography (T. Maniatis, E. Fritsch, J. Sambrook, Molecular cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982), 6.45).
  • An alternative method, the so-called "dot hybridization technique” uses radioactively labeled RNA probes for the detection of the RNAs transcribed in vitro (J. Flores et al., Lancet 1 (1983), 555-558).
  • Simple monitoring of the transcription for example by time-dependent fluorescence measurement (real-time analysis).
  • the present invention thus relates to a composition containing
  • ribozymes refers to catalytic RNA molecules with the ability to cleave other RNA molecules at phosphodiester bonds in a sequence-specific manner.
  • the hydrolysis of the target sequence to be cleaved is always initiated by the formation of a catalytically active complex consisting of ribozyme and substrate RNA. After cleavage, the hydrolyzed substrate oligonucleotide dissociates from the ribozyme; the latter is then available for further implementations.
  • ribozymes which can split phosphodiester bonds into trans, ie intermolecularly, are suitable for the purposes of the invention.
  • ribonuclease P C. Guerrier-Takada et al., Cell 44 (1983) 849-857
  • the known naturally occurring ribozymes hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme, Neurospora itochondriales VS ribozyme, group I and group II introns
  • self-cleaving or self-splicing catalysts that act in ice (intramolecular) (review article in P.
  • promoter used here relates to any DNA sequence which controls the transcription of the DNA sequence functionally linked therewith by the corresponding RNA polymerase in vivo (or in vitro) in prokaryotic or eukaryotic systems.
  • promoters are known to the person skilled in the art and include, for example, PolII promoters, SP6, T3 and T7 promoters.
  • Endogenous ribozyme expression in eukaryotic cells or cell extracts can be carried out, for example, by inserting the ribozyme-coding DNA sequence into the untranslated region of genes which are transcribed by RNA polymerase II and under the control of strongly transcribing promoters stand.
  • viral promoters such as the SV40 early promoter (F.
  • the ribozyme is a hammerhead ribozyme.
  • the ham erhead ribozyme is one of the smallest known ribozymes that catalyzes the site-specific hydrolysis of phosphodiester bonds (review article: K. Birikh et al., Eur. J. Biochem. 245 (1997) 1-16 ).
  • the ribozyme structure comprises three double-stranded regions (helices I, II and III) which flank the cleavable phosphodiester bond, and two highly conserved single-stranded sequences (0. Uhlenbeck, Nature 328 (1987), 596-600).
  • the above promoters can be regulated, ie they can be activated, for example by transcription activators, or inhibited by certain compounds.
  • Genes of eukaryotes and prokaryotes differ considerably with regard to the organization of the transcription unit (H. Ibelgaufts, Genotechnologie from A to Z, VCH Verlag Weinheim (1990), 219-223).
  • the 5 'flanking region of a eukaryotic gene is often referred to as the promoter region because it contains a number of distinct DNA sequence elements that are involved in the control of gene expression. These include the TATA box and the initiator sequence, which together form the core promoter.
  • Basal transcription is essentially regulated by the basic class II transcription activators (TF II A, B, D, E, F, H and Pol II). Analogous to basal transcription, one speaks of an activated transcription if additional regulatory elements influence the transcription. Activation takes place primarily by binding transcription activators to so-called "upstream activating sequences" (UAS). Examples of activated transcription are: SP1 binds to the SPI binding parts, CREB binds to the CRE element.
  • promoters By selecting suitable promoters, as described in more detail below, compounds which activate or inhibit transcription can be identified in a suitable test system.
  • the choice of promoter depends on the type of in vitro transcription system (e.g. yeast, HeLa or fungal cell extracts) and the RNA polymerase used.
  • the method described here can be used to measure whether the transcription is generally inhibited or activated, or remains unaffected.
  • the mechanism or the principle of influencing by a certain substance of a compound library is the subject of subsequent studies.
  • the DNA sequence encoding the ribozyme is preferably a linearized vector in which the transcription can be terminated by cleaving the template DNA downstream of the DNA encoding the ribozyme with a suitable restriction enzyme.
  • the DNA sequence encoding the ribozyme is functionally linked to a termination signal for transcription in in vivo applications.
  • termination signals are known to the person skilled in the art.
  • a general prokaryotic stop signal of transcription is a GC-rich region of certain symmetry, followed by an AT-rich sequence (A. Wu and T. Platt, Proc. Natl. Acad. Sei. 75 (1978), 5442-5446 ).
  • the DNA coding for the ribozyme is inserted into a vector which allows the inserted DNA to be multiplied in a suitable host.
  • Suitable vectors for propagation in prokaryotic or eukaryotic systems are, for example, pBR322, pNEB193, pUC18, pUC19 (Biolabs, USA.) (J. Sampson and 0. Uhlenbeck, Proc. Natl. Acad. Sci. USA 85 (1988), 1033 - 1037).
  • the ribozymes described above can be used as direct reporters for quantifying transcription rates in in vitro transcription systems.
  • the reporter RNA is generated endogenously, i. H. by transcribing the coding gene, for example from a suitable transcription vector.
  • compositions according to the invention preferably contain stabilized ribozymes, which means a longer lifespan of the ribozyme, e.g. guaranteed in in vitro transcription systems.
  • the ribozyme is transcribed together with stabilizing sequences that imitate the so-called “capping structures” and thereby increase the stability of the RNA against exonuclease degradation (Gene Therapy 4 (1996), 45-54; M. Sioud et al., J. Mol. Biol. 223 (1992), 831-835).
  • oligonucleotide substrate refers to any oligonucleotide, preferably RNA, that can be cleaved by the ribozyme, the cleaved oligonucleotide substrate being distinguishable from the uncleaved oligonucleotide substrate and producing an immediately measurable signal.
  • the substrate carries an anchor group at one end, which allows it to be immobilized on a suitable matrix, and a reporter group at its other end, which serves to detect the immobilized (uncleaved) substrate. In the absence of the ribozyme, the substrate remains intact and can be easily detected after its immobilization on the matrix, since the anchor group is still connected to the reporter group.
  • the reporter-specific signal cannot be detected, since the reporter group was separated from the anchor group due to the cleavage of the substrate.
  • the anchor group eg biotin
  • the substrate can also be immobilized via complementary sequence hybridization, provided the cleavage site and reporter group are located beyond the hybridization site. Easily detectable reporter groups that are easy to couple to nucleic acid ends are, for example, 32 P, dye molecules and molecules that can be detected with labeled antibodies.
  • the oligonucleotide substrate according to the invention is essentially complementary to the sequence (s) of the ribozyme which is (are) responsible for the substrate binding, ie it has a complementarity which allows attachment to the ribozyme in a manner which that an effective and specific cleavage of the oligonucleotide substrate is ensured.
  • the oligonucleotide substrate is preferably completely complementary to the sequences of the ribozyme which are responsible for substrate binding.
  • the length of the region of the oligonucleotide substrate which attaches to the ribozyme is preferably 5 to 8 nucleotides (P.
  • the 5 1 - and / or 3 'end of the oligonucleotide substrate may contain additional sequences which are not involved in the attachment to the ribozyme.
  • the above oligonucleotide substrate is double-labeled, the cleaved substrate being easily distinguishable from the intact substrate.
  • the in vitro transcription approach contains a terminally biotinylated substrate oligonucleotide that is labeled with fluorescein at its other end. After the minimum incubation time, which is sufficient to cleave the substrate in the case of unimpeded transcription, the transcription is stopped. The transcription batch is then incubated with a streptavidin-coated solid phase (e.g. with a commercially available microtiter plate) in order to enable the coupling of the biotinylated substrate end to the streptavidin matrix. After removing the transcription batch and washing the matrix, it is measured. With undisturbed (i.e.
  • FIG. 2b shows the Eadie-Hofstee plot for determining the ⁇ cat / K M values as a result of this measurement.
  • the kinetic parameters were determined in a conventional method with a 32 P-labeled substrate without FRET labeling (S1) (FIG. 2a).
  • the conventional method involves the separation of the cleavage products by polyacrylamide gel electrophoresis and the subsequent evaluation of the gels by autoradiography (P. Hendry, MJ McCall, TJ Lockett, Characterizing Ribozyme cleavage reactions; PC Turner (ed.); Humana Press: Totowa, NJ ( 1997), vol. 74, 221-229).
  • FIG. 3a shows the time-dependent course of the cleavage as a result of a typical measurement.
  • FIG. 3b-d shows the dependence of the reaction rate on the pH value, the temperature and the Mg 2+ concentration.
  • a pH optimum was determined at a value of 8
  • the optimum of the [Mg 2+ ] was reached at 8 mM.
  • the relatively low temperature optimum of 32 ° C probably reflects the comparatively weak binding of the
  • FIG. 4 shows the result of the measurements of a T7 RNA polymerase-dependent transcription of ribozyme-coding DNA matrices in the presence of HeLa cell nucleus extracts. As a control, the measurement was carried out in parallel in the absence of the T7 RNA polymerase and on the other hand with the inactive HHR mutant.
  • the corrected curve shows a sigmoid course, which reflects the steady increase in the ribozyme concentration in the course of the transcription reaction.
  • DNA oligonucleotides analogous to the RNA oligonucleotide substrates described above are commercially available (for example from PE Applied Biosystems, Foster City, CA, USA) or can be obtained by simple synthesis (Livak et al., PCR Methods Appl. 4 (1995) 1-6 and Rudert et al., BioTechniques 22 (1997) 1140-1145).
  • the double-labeled DNA oligonucleotides for the semiquantitative analysis of PCR amplified DNA can be used (Taqman ®, PE Applied Biosystems, Foster City, CA, USA; see also eg Lang et al, J. Immun Methods 203 (1997).. 181-192).
  • the Taqman ® -PCR technique uses the intrinsic 5 '->3' nuclease activity of the Taq polymerase enzyme: During the amplification, the 5 'and 3' double-labeled DNA oligonucleotide is hydrolyzed by the enzyme. After cleaving the DNA probe, the fluorophore and quencher diffuse apart, which results in the cancellation of the fluorescence quenching. The fluorescence of the fluorophore is then measured and serves as a measure of the amplification achieved (Livak et al., Research News (1995), PA Applied Biosysthems, Foster City, CA, USA).
  • RNA-based substrates can also be implemented without problems.
  • Methods for labeling ribonucleic acids with fluorophoric or fluorescence-quenching groups and techniques for measuring energy transfer (quenching) have already been described in detail (Turner (ed.), Ribozyme protocols, Humana press (1997), 241-251).
  • the synthetic and enzymatic production of ribozymes and the production of linearized transcription vectors are also known to the person skilled in the art (Turner (ed.), Ribozyme protocols, Humana press (1997) 51-111). 5 'fluorophore and 3' quencher labeled
  • RNA oligonucleotides as well as the analog DNA oligonucleotides are commercially available (for example 5'-FAM and 3'-TAMRA-labeled RNA from Eurogentec, Belgium).
  • the labeling is advantageously carried out at the RNA ends in order not to influence the hybridization of the ribozyme.
  • nuclease-resistant oligonucleotide substrates In order to avoid the fluorescence emission associated with undesired cleavage (for example by nucleases in the transcription system), the use of nuclease-resistant oligonucleotide substrates is particularly advantageous (Eaton and Pieken, Annu. Rev. Biochem. 64 (1995), 837-863 and Shimayama et al., Nucleic Acids Res. 21 (1993), 2605-2611). This is particularly advantageous with regard to in vivo applications in which the double-labeled substrate is introduced exogenously into cells by suitable techniques (for example microinjection, liposome transport, etc.) (P. Turner (ed.), Ribozyme protocols, Humana press (1997), 417-451).
  • the double-labeled substrates are modified RNA oligonucleotides.
  • the substrate can contain deoxyribonucleotides and / or modified bases or / and 2 '- modified ribose units. This increases the stability of the substrate in the cell extract (N. Taylor et al., Nucleic Acids Res. 20 (1992), 4559-4565).
  • the present invention further relates to a method for the quantitative determination of transcription rates, comprising the following steps:
  • Ribozyme-coding DNA sequences are known to the person skilled in the art. According to the desired ribozyme type and the appropriate oligonucleotide substrate sequence, the DNA sequence encoding the ribozyme according to the invention can be produced according to techniques known to the person skilled in the art.
  • Either double-stranded PCR-DNA or a linearized vector serves as a template for the in vitro transcription of the ribozyme.
  • Methods for producing corresponding matrices are known to the person skilled in the art (Turner (ed.), Ribozyme protocols, Humana press (1997), 69-78 and 121-139).
  • Hammerhead ribozymes are particularly preferred. Since pH, temperature, Mg 2+ concentration and the type and length of the substrate-binding sequences strongly influence the ribozyme activity, it is important to select the combination of ribozyme and substrate that results in optimized conversion rates under the physiological conditions of the respective test System leads. Suitable procedures are known to the person skilled in the art.
  • ribozyme variants and corresponding substrates can be designed (see example 1).
  • the ribozyme it is particularly important to ensure that, apart from the promoter sequence itself, the transcribed region directly following the promoter (of up to 10 nucleotides) often has a decisive influence on the transcription rates. Since the 5 'transcribed sequence is generally identical to part of the substrate hybridization area, it should be noted in particular when designing the ribozyme and substrate sequences that both optimal transcription and ribozyme-substrate hybridization are possible is. When selecting the double-marked substrate sequence, the following points must also be observed:
  • the NUH interface is advantageously GUC or AUC.
  • the substrate preferably does not form any intramolecular secondary structures.
  • the 5'-terminal base should not be guanosine.
  • the substrate should advantageously hybridize symmetrically to the ribozyme over 12 to at most 16 nucleotides.
  • An example of a suitable in vitro transcription system is the class II system from human cell nucleus extracts
  • the activity of hammerhead ribozymes is usually sufficiently high under physiological conditions, but can be increased, for example, by increasing the Mg 2+ concentration to 5 to
  • a suitable DNA template is added to the cell extract, which enables the desired ribozyme to be transcribed.
  • the oligonucleotide substrate is advantageously added to the batch in excess in order to ensure linear, reproducible dependencies between the accumulated reporter ribozyme and the measured signal (preferably a fluorescence signal) during the measurement period.
  • a parallel measurement of different transcription batches can take place, for example, in commercially available "96-well" microtiter plates. After the potential inhibitors or activators to be tested have been added, the in vitro transcription is then started, for example by adding the corresponding RNA polymerase. Suitable reaction times range from about 1 minute to about 60 minutes.
  • the transcription rate is measured by determining the fluorescence with the amount of cleaved
  • RNA 18th Oligonucleotide substrate is coupled.
  • the actual amount of transcribed RNA can be determined in appropriately calibrated systems.
  • the calibration can be carried out as follows, for example. Known amounts of ribozyme-encoding DNA are incubated with known amounts of double-labeled substrate. The transcription takes place in the presence of 32 P- ⁇ -UTP, which is incorporated into the resulting ribozyme RNA. In identical batches, the increase in fluorescence is then measured as a function of time, the individual batches being stopped at different times. To quantify the resulting amounts of ribozyme, the batches are separated on a denaturing polyacrylamide gel and evaluated with the aid of a phosphor imager. The amount of ribozyme for each measurement point can then be determined via the rate of incorporation of the radionucleotide. The calibration curve for subsequent measurements is finally obtained by plotting the measured relative fluorescence against the determined ribozyme concentration.
  • the measurement of the emitted fluorescence is advantageously carried out automatically with the aid of a suitable "read-out" device (for example ABI Prism 770 Sequence Detection System, Perkin Elmer).
  • a suitable "read-out” device for example ABI Prism 770 Sequence Detection System, Perkin Elmer.
  • Methods for calibration and the fluorescence measurement itself as well as programs for computer-aided data processing are described in detail (manual for ABI Prism 770 Sequence Detection System, Perkin Elmer; Rudert et al., BioTechniques 22 (1997) 1140-1145).
  • the fluorescence of the fluorophore is recorded over time ( ⁇ R n value) for each transcription batch as a function of time.
  • the evaluation can then be carried out on the ABI Prism 7700 device itself by adding the spectra of the different approaches
  • the present invention further relates to a method for identifying inhibitors of transcription or transcription activators, comprising the following steps:
  • the basic procedure is identical to that described above, except that not only one test system is used, but two systems which differ only in one point, namely in that the second test system contains the compound to be examined.
  • the present invention thus also relates to the composition described above which contains a DNA sequence which encodes an in vitro-selected ribozyme.
  • the present invention relates to the use of the composition described above for the absolute or comparative measurement of transcription rates, preferably by means of the methods described above.
  • the invention further relates to the use of the methods according to the invention for measuring the catalytic activity of ribozymes.
  • kits containing the compositions described above preferably for carrying out the methods according to the invention.
  • HHRlmut-DNA + T7 RNA polymerase HHRl-DNA without T7 RNA polymerase
  • HHRl-DNA + T7 RNA polymerase HHRl-DNA + T7 RNA polymerase corrected for the non-specific background activities (see Example 2).
  • the ribozyme-coding DNA sequence HHR1-DNA and the RNA substrate SL1 described in this example are derived from a known hammerhead ribozyme-substrate complex (M. Fedor and 0. Uhlenbeck, Proc. Natl. Acad. Sei. USA 87 (1990 ), 1668-1672).
  • the template for the in vitro transcription of the ribozyme HHR1, the ribozyme-coding double-stranded HHR1-DNA was generated by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the sense strand of the ribozyme was included
  • a typical 100 ⁇ l reaction mixture contained: 10 mM Tris-HCl, pH 8.9, 100 mM KC1, 1.5 mM MgCl 2 , 50 mg / ml bovine serum albumin, 0.05% Tween 20 (v / v), 200 ⁇ M dA / dC / dG / dTTP, 2 ⁇ M 5 • and 3 'primer, approx. 200 nM single-stranded DNA template and 2.5 units of Tth DNA polymerase.
  • the amplification was carried out according to the manufacturer's protocol (Boehringer Mannheim, Tth DNA polymerase kit) in four to five PCR cycles at 95 ° C, 55 sea; 55 ° C, 1 min .; 72 ° C, 1 minute.
  • the amplified DNA was then isolated by standard methods and purified from excess primers on an agarose gel (T. Maniatis, E. Fritsch, J. Sambrook, Molecular cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982), 14.2- 14.35).
  • a double-stranded DNA template coding for an inactive ribozyme variant HHRlmut, was produced starting from the following single-stranded DNA: 5'- TCT AAT ACG ACT CAC TAT A GGG TCC TCT TAG GAG GCC GTT AGG CCA GAA CTC GT-3 '(HHRlmut; primer binding sites are in italics, mutations are highlighted).
  • the following primers 5 '-TCT AAT ACG ACT CAC TAT A-3 ' (5 'primer) and 3'-GG CAA TCC GGT CTT GAG CA-5' (3 'primer) were used.
  • the 5'-FAM and 3 '-TAMRA-labeled substrate RNA SL1 with the sequence 5'-FAM-ACG AGU CAG GAU U-TAMRA-3' was obtained from Eurogentec (Belgium) and purified using a 20% denaturing polyacrylamide gel (P. Turner (ed.), Ribozyme protocols, Humana press (1997) 79-81).
  • a typical 50 ⁇ l reaction mixture contained: 40 mM TrisHCl, pH 8.0, 50 mM NaCl, 2 mMsper idin, 5 mM dithiothreitol, 5 - 25 mM MgCl 2 , approx. 500 nM SL1, 0.2 - 2 ⁇ M HHRl-DNA resp HHRImut DNA, 4 mM A / C / G / UTP and 50 units of T7 RNA polymerase. It is important to ensure that only double-stranded DNA is used in the transcription, since both single-stranded sense-strand DNA and 3 'primer DNA could inhibit the cleavage reaction.
  • the in vitro transcription was started by adding the polymerase and it was incubated at 37 ° C. for the duration of the measurement.
  • a typical 50 ⁇ l reaction batch for in vitro transcription for ribozyme production contained: 40 mM Tris-HCl, pH 8.0,
  • RNA samples 50 mM NaCl, 2 mM spermidine, 5 mM dithiothreitol, 8 mM MgCl 2 , 0.2 - 2 ⁇ M HHRl-DNA or HHRlmut-DNA, 4 mM A / C / G / UTP, 40 units of RNAsin (Promega, Madison, WI) and 50 units of T7 RNA polymerase (Stratagene, Heidelberg).
  • the RNA was purified using a 16% denaturing polyacrylic gel (37.5: 1).
  • the mixture additionally contained 100 nM SLl and 10 units of HeLa core extract (HeLa Cell Extract Transcription System, Promega, Madison, WI).
  • the in vitro transcription was started by adding the polymerase and core extract and incubated at 37 ° C. for the duration of the measurement.
  • the real-time measurements of the increase in fluorescence due to ribozyme were carried out in 50 ⁇ l reaction batches.
  • the measured signal includes both the chemical step of the cleavage and the release of the cleavage products.
  • the proportion of cleaved substrate RNA relative to the negative control was then determined by plotting the relative fluorescence of the fluorophore ( ⁇ R n value) at 535 nm over time.
  • the raw data was imported into Microsoft Excel, processed and then evaluated using the KaleidaGraph program (Abelbeck Software, Kunststoff).

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Abstract

L'invention concerne des compositions (systèmes rapporteurs) qui contiennent une séquence d'ADN codant pour un ribozyme et un substrat oligonucléotidique pouvant être clivé par le ribozyme transcrit par ladite séquence d'ADN. Dans l'un des modes de réalisation préférés, le substrat oligonucléotidique est marqué avec un groupe fluorogène (groupe rapporteur) et un groupe extincteur de fluorescence (groupe extincteur). Après clivage par le ribozyme, l'extinction de la fluorescence du fluorogène par les groupes extincteurs est interrompue et un signal de fluorescence est généré. L'invention concerne également une technique qui permet de mesurer la vitesse de transcription, par exemple afin d'identifier des substances inhibitrices de la transcription ou des activateurs de la transcription.
EP99915650A 1998-03-17 1999-03-17 Composition contenant un adn codant pour un ribozyme et un substrat oligonucleotidique, et technique permettant de mesurer la vitesse de transcription Withdrawn EP1064405A1 (fr)

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DE19811618A DE19811618C1 (de) 1998-03-17 1998-03-17 Ribozym codierende DNA und ein Oligonucleotidsubstrat enthaltende Zusammensetzung und Verfahren zur Messung von Transkriptionsraten
DE19811618 1998-03-17
PCT/EP1999/001776 WO1999047704A1 (fr) 1998-03-17 1999-03-17 Composition contenant un adn codant pour un ribozyme et un substrat oligonucleotidique, et technique permettant de mesurer la vitesse de transcription

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US6451535B1 (en) 2002-09-17
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US6861223B2 (en) 2005-03-01
WO1999047704A1 (fr) 1999-09-23
US20030008315A1 (en) 2003-01-09

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