2′-fluoropyrimidine anti-calf intestinal phosphatase nucleic acid ligands

#Described herein are high affinity nucleic acid ligands to calf intestinal phosphatase (CIP). Also described herein are methods for identifying and preparing high affinity nucleic acid ligands to CIP. The method used herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. Further disclosed are RNA ligands to CIP. Also included are oligonucleotides containing nucleotide derivatives chemically modified at the 2′-positions of pyrimidines. Additionally disclosed are RNA ligands to CIP containing 2′-F modifications. The invention also includes high affinity nucleic acid inhibitors of CIP. The oligonucleotides of the present invention are useful as diagnostic agents.

#Calf intestinal alkaline phosphatase (CIP) is a commonly used reporter enzyme for research and clinical assays. These assays typically use synthetic substrates that become detectable upon removal of phosphate groups by CIP. For example, one commonly used substrate is 1,2 dioxetane. This substrate becomes chemiluminescent upon the removal of phosphate groups by CIP. Another common substrate is the chromagen p-nitrophenylphosphate. Antibodies conjugated to CIP are widely used in ELISA, and nucleic acid probes linked to CIP can be used in a variety of nucleic acid detection schemes. Given the widespread use of CIP in various target detection schemes, it would be desirable to provide a class of ligands distinct from antibodies to which CIP could be conjugated.

#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,” and U.S. Pat. No. 5,270,163 (see also WO 91/19813), entitled “Methods for Identifying Nucleic Acid Ligands,” each of which is specifically incorporated by reference herein in its entirety. 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.

#FIGS. 1A-1D illustrate nucleic acid ligand sequences obtained from the round 8 SELEX pool.

#FIG. 2 illustrates CIP binding isotherms for the determination of the equilibrium dissociation constant at 37° C. for random 2′-F RNA (black squares) and the round 8 SELEX pool (black circles). Data are shown as the mean+/−the SEM (standard error margins) for duplicate determinations at each point.

#FIG. 3 depicts CIP binding isotherms for the determination of the equilibrium dissociation constant at 37° C. for individual clones from the round 8, SELEX pool. Data are shown as the mean+/−the SEM for duplicate determinations at each point.

#The central method utilized herein for identifying nucleic acid ligands to CIP is called the SELEX process, an acronym for Systematic Evolution of Ligands by Exponential enrichment and involves: a) contacting the candidate mixture of nucleic acids with calf intestinal phosphatase, or expressed domains or peptides corresponding to calf intestinal phosphatase, b) partitioning between members of said candidate mixture on the basis of affinity to calf intestinal phosphatase, and c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to calf intestinal phosphatase.

#Definitions

#Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided: