Modified SELEX processes without purified protein

Details Modified SELEX processes without purified protein Modified SELEX processes without purified protein

2000-09-22

2002-04-23

Zitomer, Stephanie W. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S005000, C435S007100, C435S029000, C435S091200, C435S071200, C530S300000, C530S350000, C536S022100, C536S023100, C536S025400

Reexamination Certificate

active

06376190

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed toward a method for obtaining nucleic acid ligands against target proteins without directly purifying the target proteins. The method used in the invention is called the SELEX process, which is an acronym for Systematic Evolution of Ligands by EXponential enrichment.
BACKGROUND OF THE INVENTION
The past ten years have seen phenomenal advances in the characterization of the genomes of many species. Indeed, the human genome sequence—encoding for approximately 100,000 proteins—is now substantially complete. With the completion of a genome sequence, the linear amino acid sequences of all the proteins potentially encoded by that genome are known. The goal of the biomedical research community is to use the genomic data to learn about the functions of the proteins that are encoded by the genome, and then determine the role that these proteins play in pathogenesis and disease. Unfortunately, the tools for identifying the function of proteins—their structural or enzymatic activities, and their level of synthesis—are dramatically less well developed than those for determining genomic sequences. As a result, the characterization of the functions of such proteins is the rate limiting step in the exploitation of genomic data for the development of new diagnostic and therapeutic agents.
Although some proteins are identified solely through the existence of their coding sequence in the genome, more functional approaches to protein identification and characterization have been devised. For example, one approach involves isolating all the proteins that are expressed under predetermined conditions in a certain tissue, then resolving those proteins from one another by electrophoresis on a 2-dimensional gel. Following separation, individual protein “spots” on the gel are picked and proteolytically-digested to yield peptides. The resulting peptides can be analyzed by reiterative mass spectrometry in order to determine their (partial) linear amino acid sequences. Finally, the amino acid sequences of the peptides are used to search genomic or cDNA sequences in order to obtain the DNA sequence that encodes the protein from which the peptide was derived. In this way, it is possible to prepare protein and gene expression profiles. However, because this approach is extremely labor and capital-intensive—requiring several days to analyze a single gel—it is not suited to high-throughput, routine diagnostic applications.
Regardless of the manner in which a protein implicated in disease is initially identified, it is ultimately crucial to obtain ligands to that protein, because such ligands can serve as therapeutic or diagnostic reagents. In order to generate ligands, it is necessary to have a purified source of the protein. However, because important proteins are often present in vanishingly-small amounts in biological tissues, purification—if it is even possible at all—is often a costly, labor-intensive, and time-consuming procedure. Expression of proteins is also fraught will difficulties, often because of the complexity of the post-translational modifications seen in mammalian proteins. Because of these difficulties, there is a need in the field of functional genomics for a method of generating ligands of target proteins without first requiring that the target protein be directly purified.
There have been several attempts in the art to overcome these difficulties by generating ligands of synthetic peptides with the same linear amino acid sequence as a portion of the target protein. The hope in this approach is that the ligand—typically an antibody—to the peptide will recognize the same peptide in the natural context of the intact protein. There are two fundamental problems with this approach. First, because protein structures have a large internal mass compared to their external surface, most peptide sequences from a specific protein lie within the internal mass of the protein and are not exposed to solvent. As a result, many ligands to peptides will not be able to access the same peptides within the intact protein. Second, isolated peptides typically have random, undefined structures, whereas the same peptide in the intact protein will have one or a few defined structures as a result of intramolecular constraints imposed upon it. Because ligands are generated using the isolated peptide as the target, many ligands will not recognize the defined peptide structure within the intact protein. Both of these problems cause anti-peptide antibodies to have weak affinities for the proteins that contain the same peptides.
A new class of non-protein-based ligands is found in nucleic acid molecules. The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands”, U.S. Pat. No. 5,270,163 (see also WO91/19813) entitled “Nucleic Acid Ligands” each of which is specifically incorporated by reference herein. Each of these patents and applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic a acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, 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 specifically 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 highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” describe the use of the SELEX process in conjunction wi

Affiliated with

Also associated with

Rating

Modified SELEX processes without purified protein does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Modified SELEX processes without purified protein, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Modified SELEX processes without purified protein will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2870107