1
NUCLEIC ACID LIGAND DIAGNOSTIC
BIOCHIP
CROSS REFERENCE TO RELATED
APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 09/581,465, filed Aug. 14, 2000, which is a 35 U.S.C. §371 national phase application of International Application No. PCT/US98/26515, published as International Publication No. WO 99/31275, each of which is entitled "Nucleic Acid Ligand Diagnostic Biochip." PCT/ US98/26515 is a continuation-in-part of U.S. patent application Ser. No. 08/990,436, filed Dec. 15, 1997, now U.S. Pat. No. 6,242,246, entitled "Nucleic Acid Ligand Diagnostic Biochip."
FIELD OF THE INVENTION
The invention is directed to methods for the detection of target molecules in test solutions, particularly medically relevant molecules contained in bodily fluids. The methods described herein use specific nucleic acid ligands attached to solid supports at spatially discrete locations. The invention provides methods for detecting the binding of target molecules to nucleic acid ligands, and methods for using arrays of nucleic acid ligands in diagnostic medical applications.
BACKGROUND OF THE INVENTION
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by Exponential Enrichment, termed the SELEX process, is described in U.S. patent application Ser. No. 07/536, 428, 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, U.S. patent application Ser. No. 07/931,473, filedAug. 17,1992, entitled "Methods for Identifying Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX patent applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
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 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 acidtarget 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.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such
2
as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing
5 modified nucleotides are described in U.S. patent application Ser.No. 08/117,991, filed Sep. 8,1993, abandoned in favor of U.S. patent application Ser. No. 08/430,709, filed Apr. 27, 1995, now U.S. Pat. No. 5,660,985, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,"
10 that describes oligonucleotide containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, abandoned in favor of U.S. patent application Ser. No. 08/443,957, filed May 18, 1995, now U.S. Pat. No.
15 5,580,737, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-0-methyl (2'-OMe). U.S. patent
20 application Ser. No. 08/264,029, filed Jun. 22, 1994, now abandoned, entitled "Novel Method of Preparation of Known and Novel 2' Modified Nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotide containing various 2'-modified pyrimidines.
25 Given the remarkable ability of nucleic acid ligands to be generated against many different target molecules, it would be desirable to have methods for using said ligands as a diagnostic tool. In particular, it would be desirable to attach a plurality of different nucleic acid ligands to a micro fabricated
30 solid support (a "biochip"), and then assay the binding to said ligands of target molecules in a bodily fluid. The subject application provides such methods.
SUMMARY OF THE INVENTION
35
Methods are provided in the instant invention for obtaining diagnostic and prognostic nucleic acid ligands, attaching said ligands to a biochip, and detecting binding of target molecules in a bodily fluid to said biochip-bound nucleic acid
40 ligands. In one embodiment of the instant invention, one or more nucleic acid ligands are chosen that bind to molecules known to be diagnostic or prognostic of a disease; these ligands are then attached to the biochip. Particular methods for attaching the nucleic acid ligands to the biochip are
45 described below in the section entitled "Fabrication of the Nucleic Acid Biochip." The biochip may comprise either (i) nucleic acid ligands selected against a single target molecule; or more preferably, (ii) nucleic acid ligands selected against multiple target molecules. In the subject invention, the level
50 of target molecule binding to nucleic acid ligands at defined spatial locations will be determined using bodily fluid from individuals known to have the disease for which that target molecule is known to be prognostic or diagnostic, and also using bodily fluid from healthy individuals. Bodily fluid from
55 an individual seeking a prognostic report can then be assayed using the biochip, and comparison of the three sets of data will yield prognostic or diagnostic information for that individual.
In another embodiment, the specific nucleic acid ligands 60 attached to the biochip bind specifically to all or a large number of components of blood plasma, or other bodily fluids, of a healthy individual. The pattern and level of binding of these ligands to their targets will then be determined by the methods disclosed below for healthy individuals, and also for 65 individuals diagnosed with various medical conditions. A computer database of biochip binding data will then be established, with each disease giving rise to a unique "signature"
