Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-20
2002-09-17
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023200
Reexamination Certificate
active
06451535
ABSTRACT:
The present invention relates to compositions (reporter systems) containing a DNA sequence encoding a ribozyme, preferably a hammerhead ribozyme, and a oligonucleotide substrate which is cleaved by the ribozyme transcribed by the DNA sequence. In a preferred embodiment, a FRET oligonucleotide is used, i.e. an oligonucleotide substrate which is labeled with a fluorophore group (reporter group) and a fluorescence-quenching group (quenching group), wherein the fluorescence of the fluorophore is prevented from being quenched by the fluorescence-quenching group after cleavage by the ribozyme has taken place, and therefore a fluorescence signal is generated. Moreover, the present invention relates to methods for measuring-transcription rates, for instance for determining transcription inhibitors or transcription activators, with the use of the composition of the invention, and methods for measuring the catalytic activity of ribozymes.
As a rule, the mechanisms of eukaryotic and prokaryotic transcription are examined by methods by which the mRNA is synthesized in vitro (in vitro transcription) in a cell free system, that is to say with the use of correspondingly processed cell extracts. For the production of transcripts, specifically developed transcription vectors which also carry the promoter for a corresponding RNA polymerase in addition to the reporter gene are used in this process. For instance, if the influence of particular transcription activators on the transcription is to be examined, the mRNA of the coding reporter gene must be detectable and quantifiable with suitable methods. For this purpose, radioactively labeled nucleoside triphosphates which are incorporated into the resulting mRNA, are in general added to the cell extract. 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 to detect the in vitro transcribed mRNAs (J. Flores et al., Lancet 1 (1983), 555-558). In principle, there also exists the possibility of detecting transcription indirectly via the activity of the luciferase enzyme, wherein the transcription of the luciferase reporter gene is coupled to an in-vitro translation system. The disadvantages of the above-described and partly routinely used assay methods are in particular the use of larger amounts of radioactively labeled nucleoside triphosphates and/or the high amount of methodical work and time involved in the quantification of the RNA transcripts. More suitable methods for a direct, fast and sensitive measurement of transcription rates are, however, of interest not only for scientific questions, but also for the biotechnological industry. For instance, in the so-called high throughput screening combinatory substance libraries are screened for potential guiding structures which influence the transcription of particular therapeutically relevant target genes. For these and similar applications too, a technically simple, sensitive assay system would constitute a great progress, as there are no satisfactory solutions so far.
Thus, the present invention is based on the problem of providing methods and systems suitable for these methods which permit a simple and sensitive measurement of transcription rates.
This technical problem is solved by the embodiments characterized in the patent claims. It has been found that the composition of the invention (reporter system) surprisingly allows the above-mentioned problems to be circumvented. This system has the following advantages inter alia:
Direct detection of the mRNA in the cell extract.
Fast, reproducible and technically simple quantification of transcription rates, for instance via automated fluorescence measurement.
Highly sensitive and highly specific detection of even the smallest amounts of RNA transcripts (catalytic principle for signal intensification).
Simple control of the course of transcription, for instance by time-dependent fluorescence measurement (real time analytics).
Thus, the present invention relates to a composition containing
(a) a ribozyme-encoding DNA sequence which is operatively linked to a promoter and/or regulatory elements; and
(b) an oligonucleotide substrate which is cleaved by the ribozyme transcribed by the DNA from (a),
with a directly measurable signal being generated after cleavage, as the cleaved oligonucleotide substrate can be distinguished from the non-cleaved oligonucleotide substrate.
The expression “ribozymes” as used herein relates to catalytic RNA molecules capable of cleaving other RNA molecules at phosphodiester bonds in a manner specific to the sequence. Here, 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 reactions.
In principle, all ribozymes capable of cleaving phosphodiester bonds in trans, that is to say intramolecularly, are suitable for the purposes of the invention. Apart from 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 mitochondrial VS ribozyme, group I and group II introns) are catalysts, which, however, cleave or splice themselves and which act in cis (intramolecularly) (review article in P. Turner (editor), Ribozyme protocols, Humana press (1997), 1-9). By separating the catalytic unit from the sequence containing the cleavage site it was in all cases possible to obtain ribozyme variants cleaving in trans: the hammerhead ribozyme (J. Haselhoff and W. Gerlach, Nature 334 (1988), 585-591); hairpin ribozyme (A. Hampel and R. Tritz, Biochemistry 28 (1989), 4929-4933); the hepatitis delta ribozyme (M. Been, Trends Biochem. Sci. 19 (1994) 251-256): the Neurospora mitochondrial VS ribozyme (H. Guo et al., J. Mol. Biol. 232 (1993) 351-361); the group I intron from tetrahymena (Zaug et al., Nature 324 (1986) 429-433; the group 11 intron (S. Augustin et al., Nature 34 (1990) 383-386).
The term “promoter” as used herein relates to any DNA sequence by which the transcription of the DNA sequence operatively linked thereto is controlled via the corresponding RNA polymerase in vivo (or in vitro) in prokaryotic or eukaryotic systems. Such promoters are known to a skilled person and for instance include PolII promoters, SP6, T3 and T7 promoters. The endogenous ribozyme expression in eukaryotic cells or cell extracts can, for instance, be carried out by insertion of the ribozyme-encoding DNA sequence into the non-translated region of genes which are transcribed by the RNA polymerase II and are under the control of highly transcribing promoters. Examples include viral promoters, such as the early SV40 promoter (F. Cameron and P. Jennings, Proc. Natl. Acad., Sci., USA 86 (1989) 9139-9143), the promoter of the actin gene (N. Sarver et al., Science 247 (1990) 1222-1225) or a retroviral long terminal repeat, such as HIV-LTA (Koizumi et al., Gene 117 (1992), 179-184).
In a preferred embodiment of the invention, the ribozyme is a hammerhead ribozyme. The hammerhead ribozyme measuring only about 30 nucleotides in length is one of the smallest known ribozymes catalyzing 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 areas (helices I, II and III), flanking the cleavable phosphodiester bond, and two highly conserved single-stranded sequences (O. Uhlenbeck, Nature 328 (1987), 596-600). By separating the catalytic core sequence from a sequence containing the cleavage site it was possible to prepare ribozyme v
Famulok Michael
Jenne Andreas
Birch Stewart Kolasch & Birch, LLP.
Horlick Kenneth R.
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