Photoselection of nucleic acid ligands

Details Photoselection of nucleic acid ligands Photoselection of nucleic acid ligands

2000-07-19

2002-10-01

Zitomer, Stephanie (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S022100, C536S023100, C536S024300, C536S025400

Reexamination Certificate

active

06458539

ABSTRACT:

FIELD OF INVENTION
Described herein are methods for identifying and preparing high-affinity nucleic acid ligands and nucleic acid ligands capable of photocrosslinking to target molecules specifically disclosed as nucleic acid ligands to basic Fibroblast Growth Factor
155
(bFGF
(155)
). The method utilized herein for identifying such ligands is called PhotoSELEX, an acronym for Photochemical Systematic Evolution of Ligands by EXponential enrichment. This invention includes high-affinity DNA ligands capable of photocrosslinking bFGF
(155)
. Specifically disclosed are two modified ssDNA ligands to bFGF
(155)
which exhibited high sensitivity for bFGF
(155)
comparable to that of commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF
(155)
under prevailing test conditions. Additionally, the ligands were able to distinguish bFGF
(155)
from consanguine proteins, Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF) and from other proteins in serum. Further included within the scope of this invention is a method for determining the exact position of the photocrosslink between the nucleic acid ligand and the target molecule. The present invention includes the use of nucleic acid ligands capable of photocrosslinking to targets as diagnostic reagents.
BACKGROUND OF THE INVENTION
Effective diagnostics capable of early and accurate detection of marker proteins and other analyte molecules is an area of research emphasis in the pharmaceutical industry. Despite the intensity of recent efforts, many diagnostic protocols still rely on immunochemical detection techniques first described in the early 1970's (Engvall and Perlman (1971) Immunochem. 8:871-874; Engvall and Perlman (1972) J. Immunol. 109:129-135; Hoffman (1973) J. Allergy Clin. Immunol. 5:303-307; Ljungstrom et al. (1974) Parasitology 69:xxiv). The major technique to evolve from these original investigations is the enzyme-linked immunosorbent assay (ELISA) with the sandwich immunoassay protocol representing the state of the art for large molecule (e.g. protein) detection.
While the sandwich ELISA has been a reliable mainstay for protein and antigen detection, it is often a costly, time consuming and labor intensive technique. Despite efforts to expand and automate the ELISA assay, no system is currently available for the detection of a wide array of important marker proteins in a patient from a single, small sample of the patient's serum or plasma. In the present research, an in vitro selection methodology called Photochemical Systematic Evolution of Ligands by Exponential Enrichment (photoSELEX) has been employed to identify oligodeoxynucleotide ligands which may supplant antibodies evolved through immunological techniques as diagnostic agents.
Selex
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 071536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096 and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for Identifying Nucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
Since its conception more than ten years ago, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) has proven to be a valuable combinatorial technique. The SELEX methodology has been used to successfully identify natural, as well as, modified RNA and ssDNA ligands capable of binding with high affinity and specificity to an impressive array of target molecules (Ellington and Szostak (1990) Nature 346:818-822; Gold et al. (1995) Annu. Rev. Biochem. 64:763-799; Morris et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:2902-2907; Osborne and Ellington (1997) Chem. Rev. 97:349-370; Ruckman et al. (1998) J. Biol. Chem. 273:20556-20567; Schneider et al. (1995) Biochemistry 34:9599-9610; Tuerk and Gold (1990) Science 249:505-510)).
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” now abandoned (see U.S. Pat. No. 5,707,796), describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA (See U.S. Pat. No. 5,707,796). U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” now abandoned (see U.S. Pat. No. 5,763,177), describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,” now abandoned (see U.S. Pat. No. 55,580,737), describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed “Counter-SELEX.” U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,” now abandoned, (see U.S. Pat. No. 5,567,588) and U.S. patent application Ser. No. 08/792,075, filed Jan. 31, 1997, entitled “Flow Cell SELEX,” now U.S. Pat. No. 5,861,254, describe SELEX-based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” now U.S. Pat. No. 5,496,938, describes methods for obtaining improved nucleic acid ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX,” now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled “High Aff

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