Nucleic acid molecules capable of distinguishing the isoforms PrPc and PrPSc of prion proteins and processes for their production

Abstract

The invention describes a process for the identification and isolation of nucleic acid molecules capable of distinguishing the isoforms PrP c and PrP Sc of prion proteins as well as nucleic acid molecules obtainable by this process. Furthermore, pharmaceutical compositions and diagnostic compositions are described which comprise nucleic acid molecules specifically binding prion protein isoforms as well as diagnostic methods using such molecules.

1 . A process for the identification and isolation of nucleic acid molecules which are capable of distinguishing between the isoforms PrP c  and PrP Sc  of prion proteins associated with transmissible spongiform encephalopathies comprising the steps of 
 (i) incubating a prion protein isoform or peptide fragment or derivative of this prion protein isoform with a pool of nucleic acid molecules comprising different sequences;    (ii) selecting and isolating those nucleic acid molecules which are capable of binding to said prion protein isoform or fragment or derivative thereof;    (iii) optionally, amplifying the isolated nucleic acid molecules and repeating steps (i) and (ii); and    (iv) determining the binding specificity of the isolated nucleic acid molecules for the PrP c  and PrP Sc  isoforms of prion proteins.    
     
     
         2 . The process according to  claim 1 , wherein the nucleic acid molecule is RNA.  
     
     
         3 . The process according to  claim 1  or  2 , wherein the pool of nucleic acid molecules is a randomized RNA pool.  
     
     
         4 . The process according to  claim 3 , wherein the pool of nucleic acid molecules is the RNA pool M 111.1 as described in Famulok (J. Am. Chem. Soc. 116 (1994), 1698-1706).  
     
     
         5 . The process according to  claim 1 , wherein the nucleic acid molecule is DNA.  
     
     
         6 . The process according to  claim 5 , wherein the DNA is single stranded DNA.  
     
     
         7 . The process according to  claim 5 , wherein the DNA is double stranded DNA.  
     
     
         8 . The process according to any one of  claims 5  to  7 , wherein the pool of nucleic acid molecules is a randomized DNA pool.  
     
     
         9 . The process according to any one of  claims 1  to  8  wherein the transmissible spongiform encephalopathy is Scrapie, bovine spongiform encephalopathy (BSE), Creutzfeld-Jacob Disease (CJD), Gerstmann-Sträuβler-Scheinker-Syndrome (GSS), Kuru, fatal familial insomnia (FFI) or transmissible minc encephalopathy (TME), feline spongiform encephalopathy (FSE) or chronic wasting disease (CWD).  
     
     
         10 . The process according to any one of  claims 1  to  9 , wherein the prion protein isoform or fragment or derivative thereof is immobilized.  
     
     
         11 . The process according to any one of  claims 1  to  9 , wherein the prion protein isoform or fragment or derivative thereof is in solution.  
     
     
         12 . The process according to any one of  claims 1  to  11 , wherein the prion protein isoform is the isoform PrP Sc  or a fragment or derivative of this isoform.  
     
     
         13 . The process according to  claim 12 , wherein the prion protein is the derivative PrP27-30 or a fragment thereof  
     
     
         14 . The process according to any one of  claims 1  to  11 , wherein the prion protein isoform is the isoform PrP c  or a fragment or derivative of this isoform.  
     
     
         15 . The process according to  claim 14 , wherein the prion protein is the processed form PrP c 23-231.  
     
     
         16 . The process according to any one of  claims 1  to  15 , wherein the prion protein is a recombinant protein.  
     
     
         17 . The process according to  claim 16 , wherein the prion protein is part of a fusion protein.  
     
     
         18 . The process according to  claim 17 , wherein the prion protein is part of a fusion protein with oligohistidine, calmoduline binding protein, S-peptide, FLAG, green-fluorescent protein, BTag, maltose binding protein or glutathione-S-transferase.  
     
     
         19 . A nucleic acid molecule obtainable by the process according to any one of  claims 1  to  18 .  
     
     
         20 . The nucleic acid molecule according to  claim 19  which specifically binds to an isoform of a prion protein as defined in any one of  claims 10  to  15  or to a fragment or derivative thereof.  
     
     
         21 . The nucleic acid molecule according to  claim 19  or  20  which is an RNA molecule.  
     
     
         22 . The nucleic acid molecule according to  claim 19  or  20  which is a DNA molecule.  
     
     
         23 . The nucleic acid molecule according to any one of  claims 19  to  22  which comprises four stretches of three consecutive guanosine residues separated by regions between four and seven nucleotides long.  
     
     
         24 . The nucleic acid molecule according to  claim 23  which comprises a nucleotide sequence as depicted in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.  
     
     
         25 . The nucleic acid molecule according to any one of  claims 19  to  23 , which comprises a nucleotide sequence as depicted in any one of SEQ ID NO: 1 to SEQ ID NO: 13.  
     
     
         26 . The nucleic acid molecule according to any one of  claims 19  to  22  which comprises a nucleotide sequence as depicted in SEQ ID NO: 18.  
     
     
         27 . The nucleic acid molecule according to any one of  claims 19  to  26  which is modified at one or more positions in order to increase its stability and/or to alter its biophysical and/or biochemical properties.  
     
     
         28 . A pharmaceutical composition comprising a nucleic acid molecule according to any one of  claims 19  to  27  and optionally a pharmaceutically acceptable carrier.  
     
     
         29 . A diagnostic composition comprising a nucleic acid molecule according to any one of  claims 19  to  27 .  
     
     
         30 . A method for the in-vitro diagnosis of a transmissible spongiform encephalopathy, wherein at least one of the nucleic acid molecules according to any one of  claims 19  to  27  is incubated with a probe and the interaction between the nucleic acid molecules and the PrP c  or PrP Sc  isoforms of a prion protein or fragment or derivative of these isoforms is determined.  
     
     
         31 . The method according to  claim 30  wherein at least one of the nucleic acid molecules according to any one of  claims 19  to  27  is used to quantitatively determine the amount of at least one isoform of a prion protein or a fragment or derivative thereof in a probe.  
     
     
         32 . The method according to  claim 31 , wherein nucleic acid molecules are used which specifically bind to the cellular isoform PrP c  or fragment or derivatives thereof in combination with nucleic acid molecules which specifically bind to the isoform PrP Sc  or fragment or derivatives thereof and the absolute and/or relative amount of the isoforms PrP c  and PrP Sc  in the probe is determined.  
     
     
         33 . The method according to any one of  claims 30  to  32 , wherein the probe is from an organ tissue.  
     
     
         34 . The method of  claim 32  wherein the organ tissue is from brain, tonsils, ileum, cortex, dura mater, Purkinje cells, lymphnodes, nerve cells, spleen, muscle cells, placenta, pancreas, eyes, backbone marrow or peyer'sche plaque.  
     
     
         35 . The method according to any one of  claims 30  to  32 , wherein the probe is from a body fluid.  
     
     
         36 . The method according to  claim 35 , wherein the body fluid is blood, cerebrospinal fluid, milk or semen.  
     
     
         37 . A chemical compound other than a nucleic acid molecule based on information derived from a three dimensional structure of nucleic acid molecules according to any one of  claims 19  to  27  selected from the group consisting of inorganic or organic compounds.  
     
     
         38 . The chemical compound according to  claim 37 , which is a sugar, an amino acid, a protein or a carbohydrate.