Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-07
2001-01-30
Elliott, George C. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C536S023100, C536S024300, C536S025320
Reexamination Certificate
active
06180348
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the synthesis and identification of oligonucleotide ligands of target molecules and uses thereof. The invention also relates to Fluorescence Activated Cell Sorter analysis, i.e., FACScan flow cytometetry, and other convenient separation means.
BACKGROUND OF THE INVENTION
SELEX, i.e., Systematic Evolution of Ligands by Exponential Enrichment, is a process of isolating oligonucleotide ligands of a chosen target molecule (see Tuerk and Gold,
Science
249:505-510, (1990), U.S. Pat. Nos. 5,475,096, 5,595,877, and 5,660,985). SELEX as described in Tuerk and Gold involves admixing the target molecule with a pool of oligonucleotides (e.g., RNA) of diverse sequences; retaining complexes formed between the target and oligonucleotides; recovering the oligonucleotides bound to the target; reverse-transcribing the RNA into DNA; amplifying the DNA with polymerase chain reactions (PCR); transcribing the amplified DNA into RNA; and repeating the cycle with ever increasing binding stringency. Three enzymatic reactions are required for each cycle. It usually takes many cycles (e.g., between 12-15 cycles) to isolate aptamers of high affinity and specificity to the target. An aptamer is an oligonucleotide that is capable of binding to an intended target substance but not other molecules under the same conditions.
Bock et al., (1990)
Nature
355:564-566, describes another approach of isolating aptamers. Bock's process is different from that of Tuerk and Gold in that only one enzymatic reaction is required for each cycle (i.e., PCR) because the nucleic acid library in Bock's method is comprised of DNA instead of RNA. The identification and isolation of aptamers of high specificity and affinity with the method of Bock et al. still requires repeated cycles in a chromatographic column.
Conrad et al., (1996)
Methods in Enzymol
. 267:336-367, describes a variety of methods for isolating aptamers, all of which employ repeated cycles to enrich target-bound ligands and require a large amount of purified target molecules.
SUMMARY OF THE INVENTION
The present invention features a simple, speedy and cost efficient method of identifying aptamers to specific target molecules. It allows for the identification and isolation of aptamers without numerous cycles of selection and amplification.
Within the scope of this invention, Applicant successfully identified and isolated synthetic single-stranded oligonucleotides of high affinity to a target molecule of interest, erythropoietin. In addition, Applicant successfully used a synthetic single-stranded oligonucleotide in an affinity matrix to purify another molecule of interest, prothrombin. The Applicant's success in these endeavors suggests a broad-spectrum applicability of the invention that is not limited to the specific biologic molecules studied herein. Those of skill in the art will appreciate that such can be applied to any molecule or compound of interest, biologic or otherwise, that has an affinity for oligonucleotide ligand sequences of the type described herein.
In a first aspect, this invention features a method that makes use of magnetic separation to identify an aptamer which specifically binds to a target molecule of interest by magnetic separation. In this method, the target molecule is conjugated to a magnetic substance. After allowing the target molecule to mix with a collection of candidate oligonucleotides under conditions suitable for complex formation, i.e., binding of target molecule with aptamer(s), magnetic force is applied to separate aptamer(s) from the rest of the candidate oligonucleotides that have little or no affinity for the target molecule. Preferably, this method contains the following steps:
a) providing a collection of candidate oligonucleotides attached to a plurality of solid supports, wherein candidate oligonucleotides of different nucleotide sequences are attached to different solid supports and all candidate oligonucleotides attached to a single solid support are of the same nucleotide sequence;
b) providing a plurality of magnetic beads covered with the target molecule;
c) admixing the collection of candidate oligonucleotides on solid supports with the magnetic beads covered with the target molecule under conditions suitable for the binding of oligonucleotide ligands to the target molecule, wherein magnetic bead conjugates complex with one or more solid supports bearing aptamers to the target molecule of interest;
d) applying magnetic force to isolate one or more solid supports attached with magnetic beads; and
e) identifying oligonucleotides present on the isolated solid supports.
A collection of candidate oligonucleotides is generated using conventional synthesis techniques. Preferably, each oligonucleotide in the collection contains both a randomized sequence as well as at least one adjacent primer sequence for amplification and/or sequencing. Candidate oligonucleotides include single-stranded and double-stranded RNA or DNA of any length. A candidate oligonucleotide may contain modified or derivatized groups known in the art, especially those identified in U.S. Pat. Nos. 5,582,981 and 5,660,985, such as analogous forms of purines and pyrimidines and analogous forms of ribose and deoxyribose.
In
FIG. 4
, the oligonucleotides shown correspond to the sequence listing as follows: F18 is SEQ ID NO. 8; F15 is SEQ ID NO. 9; F21 is SEQ ID NO. 10; F22 is SEQ ID NO. 11; F17 is SEQ ID NO. 12; F8 is SEQ ID NO. 13; F13 is SEQ ID NO. 14; and F19 is SEQ ID NO. 15.
A target molecule can be any molecule capable of forming a complex with an oligonucleotide, including, but not limited to, peptides, proteins, enzymes, antibodies, hormones, glycoproteins, polymers, polysaccharides, nucleic acids, small organic compounds such as drugs, dyes, metabolites, cofactors, transition state analogs and toxins.
Specific target molecules of interest include molecules of biological and physiological relevance in both prokaryotic and eukaryotic organisms, particularly mammals. Examples of such biologically significant molecules in mammals include, but are not limited to, erythropoietin, tissue plasminogen activator, granular colony stimulating factor (G-CSF), growth hormone (GH), endostatin (O'Reilly et al., (1997)
Cell
88:277-285), interferons, interleukins, chemokines (Shi et al., (1997)
FASEB J
. 11:1330; Bubrovsky et al., (1996)
PNAS, USA
92:700-709), enzymes such as SOD (Yoshikai et al., (1995)
Cancer Res
. 55(8) 1617-1620) and amylase, antibodies (particularly the constant “Fc” regions thereof), OKT3 (Ho et al., (1998)
Science
280:1866-1867), serum proteins (e.g., Factor VIII (Papadopulos-Eleopulos et al., (1990)
Genetica
95:35-50), Factor VIX, plasminogen, antithrombin III (Jones et al., (1992)
Br. J. Cancer
66:744-747), albumin, protein C (Griffin et al., (1993)
Blood
82:1989-93), etc.), and vaccines (e.q., HbsAg (Davis et al., (1994) Vaccine 12:1503-1509), etc.). The physiological significance of most of these, and many other molecules, may similarly be found in Goodman and Gilman's
The Pharmacological Basis of Therapeutics
, 8
th
ed., (1990) Pergamon Press, Elmsford, N.Y. Those of skill in the art will appreciate that a virtually unlimited number of other target molecules may also be used with the claimed methods.
The solid support can be anything suitable for attaching oligonucleotides, including, but not limited to, resin beads, controlled pore glass (e.g., Maskos and Southern, (1992)
Nucleic Acid Res
. 20:1679-1684), polystyrene (e.g., McCollum and Andrus, (1991)
Tetrahedron Lett
. 32:4069-4072), PEG-polystyrene (e.g., Gao et al., (1991) Tetrahedron Lett. 32:5477-5480), Teflon (e.g., Arnold, U.S. Pat. No. 5,362,866), cellulose (e.g., Crea and Horn, (1980) Nucleic Acid Res. 8:2331-2348). In a preferred embodiment, the oligonucleotides of diverse sequences are synthesized on the solid supports (e.g., Pon, (1993)
Methods Mol. Biol
. 20:465-496). In another preferred embodiment, the solid support is of a size observable under an optical microsc
Elliott George C.
Lyon & Lyon LLP
Shibuya Mark L.
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