Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-06-07
2003-10-14
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S001110, C536S022100, C536S025300, C536S025310, C536S027100
Reexamination Certificate
active
06632938
ABSTRACT:
FIELD OF THE INVENTION
The present inventions relate to novel methods for purifying oligonucleotides. More specifically, the present inventions relate to novel methods for purifying oligonucleotides wherein the oligonucleotides are precipitated from solution and isolated using physical means.
BACKGROUND OF THE INVENTION
Oligonucleotides and their analogs have been developed and used in molecular biology as probes, primers, linkers, adapters, and gene fragments in a variety of procedures. Oligonucleotides play a significant role, for example, in the fields of therapeutics, diagnostics, and research.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and humans. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides that are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for various disease states, including use as antiviral agents. Other mechanisms of action have also been proposed. For example, transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al.,
Science,
1990, 250, 997-1000; and Wu, H., et. al.,
Gene,
1990, 89, 203-209), incorporated herein by reference in its entirety.
In addition to use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using, for example, biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits that assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as
Molecular Cloning, A Laboratory Manual
, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and
Current Protocols In Molecular Biology
, F. M. Ausubel, et al., Eds., Current Publications, 1993; each incorporated herein by reference in its entirety. Such uses include, for example, synthetic oligonucleotide probes, screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of
Molecular Cloning, A Laboratory Manual
, supra. See also “DNA-protein interactions and The Polymerase Chain Reaction” in Vol. 2 of
Current Protocols In Molecular Biology,
supra; each incorporated herein by reference in its entirety.
Owing to the wide range of applications, oligonucleotides and their analogs have been customized to provide properties that are tailored for desired uses. Thus, a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include, but are not limited to, those designed to increase binding to a target strand, to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, to improve the pharmacokinetic properties of the oligonucleotide, and to modulate uptake and cellular distribution of the oligonucleotide.
Modifications to naturally occurring oligonucleotides include, for example, labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include, but are not limited to, incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2′-O-methyl ribose sugar units.
Antisense oligonucleotides also may be modified to conjugate with lipophilic molecules. The presence of the lipophilic conjugate has been shown to improve cellular permeation of the oligonucleotide and, accordingly, improve distribution of the oligonucleotide in cells. Further, oligonucleotides conjugated with lipophilic molecules are able to enhance the free uptake of the oligonucleotides without the need for any transfection agents in cell culture studies. Conjugated oligonucleotides are also able to improve the protein binding of oligonucleotides containing phosphodiester linkages. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs.
The chemical literature discloses numerous processes for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most popular processes is the phosphoramidite technique (see, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P.,
Tetrahedron,
1992, 48, 2223-2311 and references cited therein each incorporated herein by reference in its entirety), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
The ability of the acylaminoethyl group to serve as a protecting group for certain phosphate diesters was first observed by Ziodrou and Schmir. Zioudrou et al.,
J. Amer. Chem. Soc.,
85, 3258, 1963; incorporated herein by r
Arthur John Charles
Moore Max N.
Scozzari Anthony N.
Vansooy Kent
ISIS Pharmaceuticals Inc.
McIntosh III Traviss C.
Wilson James O.
Woodcock & Washburn LLP
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