Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-10
2001-04-03
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069520, C435S091100, C536S022100, C536S023100, C536S023200, C536S023500
Reexamination Certificate
active
06210888
ABSTRACT:
The present invention discloses a technique for detecting deamination of a cytidine in RNAs or DNAs (for example a single strand mRNA, or a double strand viral RNA etc.). This technique is based on knowledge of the local sequence of the RNA or DNA containing the deaminated site. This sequence is used to produce a primer oligonucleotide (DNA or RNA) just at 3′ of the deaminated site (stopping at 3′ of the deaminated site at a site such that the intermediate sequence only comprises one, two or three types of nucleotides). This primer is labelled for example at terminal 5′ or in its proximity, in particular through incorporation of modified nucleotides (biotinilated, radiolabelled, fluorescent etc.) or by chemical modification of this terminal such that said terminal may be detected by chemical, biochemical or other reaction in particular with a substrate, or by fluorescence, radioactivity detection etc. One example of said system of detection is given by the SPA® system (Scintillation Proximity Assay) coupled to DNA primers, commercially available from Amersham.
This primer is hybridised with the RNA or DNA to be assayed in the presence of Reverse Transcriptase (for RNA/DNA hybridisation), of RNA polymerase (for RNA/RNA hybridisation), or of DNA polymerase (for DNA/DNA hybridisation) and modified adenine. If the site to be assayed is deaminated (normal case: non-deaminated), one to three nucleotides are added to each pricer hybridised to a deaminated RNA or DNA. Conversely, to detect the absence of deamination of a site (normal case: deaminated), modified quanine is used (for example biotinilated, radiolabelled, fluorescent etc.).
After extending the primers they are denatured and placed in the presence of an exposure system, either modified nucleotides to be incorporated, or primer terminals so as to detect and/or quantify the incorporation of the modified nucleotides.
One example of embodiment of this system entails a primer containing one or more nucleotides digoxygenated at its 5′ terminal or in its proximity, in which the incorporated nucleotides is biotinilated. After incorporation, the incorporated primer+nucleotide unit (extended primer) is trapped in a well treated with streptavidine, which is rinsed to remove non-extended primers, and exposed using a standard ELISA technique (see FIG.
1
). The symmetric system in which the incorporated nucleotide is digoxygenated, and the biotinilated 5′ terminal can be used just as advantageously. It is also possible, instead of labelling for example the 5′ terminal of the primer used, to label the DNA or RNA being assayed which contains the nucleotide likely to be deaminated (see FIG.
2
).
The present invention also relates to a technique which can be used to detect the deamination of methylated cytidine (5mC) in RNAs or DNAs. This technique is based upon knowledge of the local sequence of the RNA or DNA containing the deaminated site. This sequence is used to produce a primer oligonucleotide (DNA or RNA) just at 3′ of the deaminated site (stopping at 3′ of the deaminated site, at a site such that the intermediate sequence only comprises one, two or three types of nucleotides (see FIG.
1
). This primer is labelled for example at the 5′ terminal or in its proximity, in particular through incorporation of one to three modified nucleotides (biotinilated, radiolabelled, fluorescent etc.) or by chemical modification of the terminal, such that said terminal may be detected by chemical, biochemical or other reaction, in particular with a substrate, or by fluorescence, radioactivity detection etc. One example of said detection system has already been cited previously.
This primer is hybridised to the RNA or DNA to be assayed in the presence of Reverse Transcriptase (for RNA/DNA hybridisation), of RNA polymerase (for RNA/RNA hybridisation), or of DNA polymerase (for DNA/DNA hybridisation) and modified adenine. If the site to be studies is deaminated (normal case: non-deaminated), one nucleotide (at least) is added to each hybridised primer. Conversely, to detect the absence of deamination of a site (normal case: deaminated), modified quanine is used (for example biotinilated, radiolabelled, fluorescent etc.).
After extending the primers they are denatured and placed in the presence of a an exposure system, either modified nucleotides to be incorporated, or primer terminals so as to detect and/or quantify the incorporation of the modified nucleotides.
One example of embodiment of this system entails a primer containing one or more nucleotides digoxygenated at its 5′ terminal or in its proximity, in which the incorporated nucleotide is biotinilated. After incorporation, the incorporated primer+nucleotide unit (extended primer) is trapped in a well treated with streptavidine, which is rinsed to remove non-extended primers, and exposed using a standard ELISA technique (see FIG.
1
). The symmetric system in which the incorporated nucleotide is digoxygenated, and the biotinilated 5′ terminal can be used just as advantageously. It is also possible, instead of labelling for example terminal 5′ of the primer used, to label the DNA or RNA being assayed containing the nucleotide likely to be deaminated.
This invention concerns a RNA or DNA deamination detection kit containing at least one of the following elements:
a) a labelled RNA or DNA primer
b) one to three labelled nucleotides
c) the reagents and mediums required to extend the primer
d) a system for detecting the incorporation of the labelled nucleotides.
The present invention discloses a quick method for screening molecules having an inhibitory effect on the deaminase activity of an enzyme acting on a specific RNA (respectively a DNA) and a device for its implementation: This method (see FIGS
1
and
2
) consists of using a RNA (or DNA respectively) substrate containing the minimum sequence around the deaminated cytosine required for the observation of normal activity of the deaminase enzyme. To this substrate is added a crude extract of cell proteins in which this activity takes place, DNA (or RNA) primers allowing the implementation of the technique described previously for the detection of deamination of a cytosine in an RNA (respectively DNA) of the assayed cytosine, and modified nucleotides (adenine for the detection of deamination, quanine for the detection of non-deamination), plus one or more molecules or products whose inhibitory effect is to be assayed.
The present invention also relates to all molecules or products having an inhibitory effect on said enzyme or associated proteins, discovered by one of the techniques described in this invention.
This invention applies in particular to the screening of molecules inhibiting the deamination activity of apobec-1, or the activity of associated proteins (Shah R R et al (1991) J. Biol. Chem. 16301-16304, Davies M S et al (1989) J. Biol. Chem. 13395-13398), which may have therapeutic use in atherosclerosis and obesity and other diseases characterized by a higher than normal plasma level of chylomicrons and/or VLDL (hyperlipidaemia, for example hypercholesterosaemia, hypertriglyceridaemia etc.) and/or by hyperglycaemia.
The deaminated cytidine being assayed here is the cytidine at position 6666 of the mRNA of apoB, the RNA sequence used as substrate containing the anchor zone of apobec-1 or of the associated proteins is that described in the literature (Shah R R et al (1991) J. Biol. Chem. 16301-16304, Davies M S et al (1989) J. Biol. Chem. 13395-13398) (called anchor sequence of the mRNA of apoB), and the protein extracts used may be derived from rat livers or other sources. The sequence used a primer for the implementation of the technique contains a number of complementary nucleotides of the 3′ sequence of site 6666 of the mRNA of apoB that is sufficient for proper hybridisation (at least 14 nucleotides).
This invention therefore concerns a technique for detecting the deamination of cytidine at position 6666 of the mRNA of apoB as previously describe
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