Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-10-16
2002-07-09
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S091100, C536S025400
Reexamination Certificate
active
06417343
ABSTRACT:
FIELD OF THE INVENTION
The invention described below is a method for targeting nucleic acids, in particular ribonucleic acid (RNA) by incorporating into the nucleic acid a halogenated nucleic acid precursor and binding to the resulting halogenated nucleic acid an antibody to the halogenated nucleic acid precursor. This method is useful for any type of nucleic acid analysis, in particular for isolating nucleic acids, and also for labelling nucleic acids, and for separating different populations of nucleic acids. For example, this method can be used to separate deoxyriboncleic acid (DNA) from RNA and to isolate pools of nucleic acids synthesized at specific times and after specific events.
BACKGROUND OF THE INVENTION
Many methods are available for isolation and separation of nucleic acids. Most such methods are based on selective extraction of nucleic acids by organic or inorganic solvents or by specific precipitation, and do not exploit the potential of immunochemical labelling technology.
Immunological labels have been used in connection with nucleic acids in that digoxygenin or biotin -conjugated nucleotides may be used to separate out nucleic acids synthesized at the time of addition of these precursors. However, the precursors are only practical for in vitro labelling of nucleic acids already isolated from cells, since they do not easily penetrate the cell membrane. In addition, such conjugated nucleotides are expensive.
Methods for labelling DNA in vivo with halogenated precursors which are then detected by immunocytochemistry are widely used to analyze cell cycle kinetics. Detection of RNA has also been accomplished by this method. But halogenated precursors in the area of isolating and separating nucleic acids have been very little exploited. Although labelled DNA has been separated by immunoprecipitation and detected by means of antibodies immobilized on plates, no other immunocytochemical methods have been attempted with DNA. With regard to RNA, immunocytochemical separation of halogenated RNA has never been performed and it is completely unknown whether such a method could be advantageously applied to RNA.
Further, the commonly available isolation and separation methods noted above cannot differentiate between nucleic acids which are synthesized in the cell at different times, e.g. at different time intervals after induction of cell differentiation, mitogenic stimulation, carcino-genesis, drug treatment, etc. The only available approaches are analysis of transcribed DNA or nuclear runoff transcription of RNA. These methods are complex, cumbersome, and costly. Labelled nucleotides described above do not penetrate the cell membrane sufficiently well to be an alternative which provides a means of analysis readily applicable in vivo.
The inventors have solved these problems by developing a new method which exploits different aspects of these otherwise unrelated methods and provides a convenient and accessible means for isolation and separation of nucleic acids, in particular RNA, which can be used with living cells. A surprising advantage of the new method is that it permits direct separation of RNA synthesized in a given time interval from RNA which existed in the cell prior to this time interval. Such a direct method has not been available until now.
SUMMARY OF THE INVENTION
Methods for manipulating nucleic acids, in particular RNA, in a rapid, simple, and convenient manner using the tools of halogenated nucleotide precursors and antibodies capable of binding to such precursors, are the basis for this invention.
This invention provides a method for labelling RNA by incubating the RNA with a halogenated nucleotide triphosphate so as to incorporate the halogenated nucleotide triphosphate into the RNA and incubating the resulting RNA with a labelled antibody which binds specifically to the halogenated nucleotide triphosphate so as to label the RNA.
Also part of this invention is a method for isolating RNA by incubating the RNA with a halogenated nucleotide triphosphate so as to incorporate the halogenated nucleotide triphosphate into the RNA and incubating the resulting RNA with an antibody which binds specifically to the halogenated nucleotide triphosphate and which is attached to a solid support, then isolating the solid support and separating the RNA from the antibody.
The methods of this invention not only isolate RNA, but effectively separate RNA from any DNA which may be present. It is also part of this invention to separate RNA from DNA by incubating the RNA and DNA with a nucleotide triphosphate and a deoxynucleotide triphosphate, where both triphosphates are halogenated using a different halogen, differentially labelling the resulting RNA and DNA by incubating with a labelled antibody which binds specifically to the nucleotide triphosphate and a labelled antibody which binds specifically to the deoxynucleotide triphosphate, and separating the differentially labelled RNA and DNA.
Another method of this invention provides for isolating RNA synthesized during a period of time by starting to incubate the RNA with a halogenated nucleotide triphosphate at the beginning of the period of time so as to incorporate the halogenated nucleotide triphosphate into RNA, stopping the incubation at the end of the period of time, then incubating the resulting RNA with an antibody which binds specifically to the halogenated nucleotide triphosphate, and finally separating the RNA from the antibody to isolate the RNA synthesized during the period of time.
It will be apparent to the skilled person that the methods of this invention have a broad applicability both in basic research and in applied technology. The following uses for the subject methods are provided as examples:
Isolation of RNA is of interest for cloning proteins of pharmaceutical interest.
Halogen labelled nucleic acids can also be prepared in vivo by growing bacteria carrying plasmids in the presence of halogenated nucleotide precursors or in vitro by polymerase catalyzed reactions. The labelled nucleic acids can then be used as nonradioactive probes in a variety of hybridization applications and detected by routine immunochemical methods using anti-halogenated nucleotide precursor antibodies.
RNA labelled by the methods of this invention can be separated on gels and transferred to filters on which various species of RNA may be detected (since RNA bases are labelled in sufficiently high density to distinguish species), allowing for comparison of the amounts and kinds of RNA present in tumor cells versus normal cells, or before and after addition of an active agent, providing information about RNA synthetic activity.
By using pulses of halogen labelled RNA precursors as described in this invention at different times after perturbation of cells or organisms by drug treatment, radiation, signals to proliferate or differentiate, the resulting temporal sequence of events may be determined. In particular, proteins whose synthesis results from perturbation may be isolated by obtaining the corresponding RNA, and used either directly or in drug screening. Halogenated RNA precursors which are distinguished by binding to different antibodies may also be used in this way.
RNA isolated by methods of this invention may be used to determine which genes are activated as a result of drug treatment and then reverse transcribed to make complementary DNA. Such DNA may be used to obtain corresponding proteins or used directly as antisense genes to turn off gene activation if desired.
DNA may be separated from RNA by the methods of this invention, which can be used to isolate from a cell perturbed by radiation or drugs both DNA in the form of damaged genes which are being repaired and mRNA produced from genes which are not damaged and therefore are actively synthesizing proteins. From these materials, antisense genes and/or proteins can be produced for use in treatment of perturbation caused by conditions such as disease or exposure.
REFERENCES:
patent: 4711955 (1987-12-01), Ward et al.
patent: 4851331 (1989-07-01), Van et al.
patent: 4978
Darzynkiewicz Zbigniew
Juan Gloria
Traganos Frank
Amster Rothstein and Ebenstein
Marschel Ardin H.
New York Medical College
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