Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-12-08
2004-12-07
Sisson, Bradley L. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C435S091300, C435S183000, C536S023100, C536S024330, C536S025300
Reexamination Certificate
active
06828127
ABSTRACT:
The subject of the invention is a method of transcription, which makes it possible to synthesize RNA strands complementary to an RNA template, as well as new RNA polymerases which make it possible to carry out this method.
The method of the invention leads to the amplification of RNA present in small quantities in a biological sample, and thus allows the detection and/or quantification of the RNA in the sample, or the sequencing of the product of amplification, in particular in the field of microbiology and virology, and more generally in the field of medical diagnosis. The method of the invention may also be used in the synthesis of RNA probes.
It is known that in microbiology and in virology, the microorganisms which it is sought to identify are often viable bacteria (therefore containing more RNA than DNA) or RNA viruses such as the HIV and HCV viruses. It is also known that in various pathologies, it is often advantageous to monitor the variations in the expression of genes, and therefore in the synthesis of messenger RNA.
It is therefore important to be able to have a simple and effective method of amplifying an RNA target.
The PCR method, which makes it possible to cyclically amplify a DNA target, uses a single enzyme but requires the production of temperature cycles, generally at three different temperatures. The PCR method may be adapted to the amplification of an RNA target by adding an additional enzymatic activity of RNA-dependent DNA polymerase, which further complicates this method.
The so-called NASBA/TMA method of amplification has the advantage of being an isothermic method, but requires the use of three enzymatic activities (RNA-dependent DNA polymerase, RNase H and DNA-dependent RNA polymerase) carried by two or three enzymes.
It is therefore desirable to be able to have a simple and automatable method of amplification of RNA, and in particular an isothermic method using only one enzyme.
To avoid the disadvantages, which have just been mentioned, of known amplification techniques, it therefore appears to be necessary to use, for the amplification of RNA, an RNA-dependent RNA polymerase activity.
Unfortunately, the known natural RNA-dependent RNA polymerases (RNAd RNAp) are not suitable for such a use because they have specific requirements as regards the RNA template, and their activity requires the presence of protein cofactors (also called auxiliary protein factors or associated protein factors).
It has now been discovered that some known DNA-dependent RNA polymerases are capable of transcribing a single-stranded RNA in the presence of a double-stranded DNA promoter. Furthermore, some of these enzymes, which are transformed by mutation, are capable of synthesizing a transcriptional product with a better yield when the template consists of RNA than when the template consists of DNA.
In the present application, the term “transcription” designates the synthesis of several strands of RNA in the presence of a polynucleotide template and of ribonucleoside triphosphates, in an appropriate reaction medium and under conditions allowing the catalytic activity of an RNA polymerase to be exerted. The transcription occurs by synthesis of a complementary or antiparallel copy of the template. The strand of the template which is copied is called the transcribed strand or the template strand. The synthesis of the RNA progresses in the 5′-3′ direction.
It is known that some RNA polymerases function under the control of a promoter. A promoter is a double-stranded nucleotide sequence recognized for the RNA polymerase and necessary for the initiation of transcription.
It should be recalled that when the template strand is linked to the promoter, the first nucleotide transcribed on the template strand, linked by its 3′ end to the 5′ end of one of the strands of the promoter, is designated by +1. The strand of the promoter which is linked to the template strand is called the antisense strand. The other strand of the promoter, which is complementary to the antisense strand, and hybridized to it, is called sense strand. The successive nucleotides which are situated on the side of the promoter, with respect to nucleotide +1, are, starting from +1, numbered −1, −2, −3, and the like.
The position −1 therefore corresponds to the 5′ end of the antisense strand of the promoter, and to the 3′ end of the sense strand. However, some authors include the nucleotide sequence corresponding to the region where the transcription starts (in particular the sequence from +1 to +6, for which a consensus sequence can generally be defined) in the definition of the sequence of the promoter.
On the template strand, the positions of the successive nucleotides copied, starting from +1, and therefore in the 3′-5′ direction, are noted +2, +3, and the like.
In the text which follows, the terms sense strand and antisense strand are generally used for the promoter itself (positions numbered negatively), and the term non-template strand is used for any strand linked to the 3′ end of the sense strand, and the term template strand for any strand linked to the 5′ end of the antisense strand or for any strand hybridized to the non-template strand. In a given polynucleotide strand, “upstream region” refers to a region situated on the side of the 5′ end, and “downstream region” a region situated on the side of the 3′ end. However, in the domain of transcription under the control of a promoter, and without taking into consideration a particular strand, “upstream” region traditionally refers to the region which, relative to position +1, is on the side of the promoter (positions indicated by negative numbers), and “downstream” region the region situated on the side of the template copied (positions indicated by positive numbers), such that the downstream direction then corresponds to the 3′-5′ direction on the template strand, and to the 5′-3′ direction on the newly-synthesized RNA strand.
The template strand is not necessarily linked to the 5′ end of the antisense strand of the promoter. However, it should, in this case, be hybridized to a complementary and antiparallel strand (non-template strand) which is itself linked by its 5′ end to the 3′ and of the sense strand of the promoter; see ZHOU W. and DOETSCH P. W.,
Biochemistry
33, 14926-14934 (1994) and ZHOU W. et al.,
Cell
82, 577-585 (1995). In such a case, the transcription may start in any position, which may range from +1 to +24, corresponding to the 3′ end of the template strand or of the part of the template strand hybridized with the non-template strand.
Compared with bacterial, eukaryotic or mitochondrial RNA polymerases, the phage RNA polymerases are very simple enzymes. The best known among them are the RNA polymerases of the T7, T3 and SP6 bacteriophages. The bacteriophage RNA polymerase has been cloned; see in particular U.S. Pat. No. 4,952,496. These enzymes are highly homologous to one another and consist of a single subunit. The natural promoters specific for the RNA polymerases of the T7, T3 and SP6 phages are well known. The sequencing of the whole genome of the bacteriophage T7 (Dunn et al.,
J. Mol. Biol.
166, 477-535 (1983)) has made it possible to define the existence of 17 promoters on the DNA of this phage. Comparison of these 17 sequences shows that 23 contiguous nucleotides situated between positions −17 and +6 relative to the site of initiation (position +1) of transcription, are highly conserved. These nucleotides are even identical in five so-called class III promoters, which are the most efficient in particular in vitro. Likewise, many promoter sequences specific for the T3 RNA polymerase also exhibit a very high homology, in particular between positions −17 and +6. Moreover, several different sequences of promoter for phage SP6 RNA polymerase have been identified and also exhibit a high homology; see Brown J. E
Arnaud-Barbe Nadége
Cheynet-Sauvion Valérie
Mallet François
Mandrand Bernard
McAllister William T.
Bio Merieux
Oliff & Berridge,PLC
Sisson Bradley L.
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