High affinity nucleic acid ligands to lectins

Abstract

This invention discloses high-affinity oligonucleotide ligands to lectins, specifically nucleic acid ligands having the ability to bind to the lectins, wheat germ agglutinin, L-selectin, E-selectin and P-selectin. Also disclosed are the methods for obtaining such ligands. This invention discloses high-affinity oligonucleotide ligands to lectins, specifically nucleic acid ligands having the ability to bind to the lectins, wheat germ agglutinin, L-selectin, E-selectin and P-selectin. Also disclosed are the methods for obtaining such ligands.

We claim:  
     
         1 . A method for identifying nucleic acid ligands and nucleic acid ligand sequences to a lectin comprising: 
 a) contacting a candidate mixture of nucleic acids with a lectin, wherein nucleic acids having an increased affinity to said lectin relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;    b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and    c) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said lectin, whereby nucleic acid ligands to said lectin may be identified.    
     
     
         2 . The method of  claim 1  further comprising: 
 d) repeating steps a), b) and c).  
 
     
     
         3 . The method of  claim 1  wherein said candidate mixture is comprised of single-stranded nucleic acids.  
     
     
         4 . The method of  claim 3  wherein said single-stranded nucleic acids are ribonucleic acids.  
     
     
         5 . The method of  claim 4  wherein said nucleic acids comprise modified ribonucleic acids.  
     
     
         6 . The method of  claim 5  wherein said nucleic acids comprise modified ribonucleic acids selected from the group consisting of 2′-amino (2′- NH 2 ) modified ribonucleic acids and 2′-fluoro (2′-F) modified ribonucleic acids.  
     
     
         7 . The method of  claim 3  wherein said single-stranded nucleic acids are deoxyribonucleic acids.  
     
     
         8 . The method of  claim 2  further comprising 
 e) forming a multivalent Complex comprising two nucleic acid ligands identified in step c).  
 
     
     
         9 . The method of  claim 5  further comprising 
 e) substituting 2′-O-methyl ribonucleic acids for 2′-OH ribonucleic acids in the nucleic acid ligands identified in step c).  
 
     
     
         10 . The method of  claim 1  wherein said lectin is selected from the group consisting of a mammalian lectin, a plant lectin, a microbial lectin and a viral lectin.  
     
     
         11 . The method of  claim 1  wherein said lectin is wheat germ agglutinin.  
     
     
         12 . The method of  claim 1  wherein said lectin is a selectin.  
     
     
         13 . The method of  claim 12  wherein said selectin is selected from the group consisting of L-selectin, E-selectin, and P-selectin.  
     
     
         14 . The method of  claim 1  wherein said lectin is serum mannose binding protein.  
     
     
         15 . A purified and isolated non-naturally occurring nucleic acid ligand to a lectin.  
     
     
         16 . The nucleic acid ligand of  claim 15  which is a non-naturally occurring nucleic acid ligand having a specific binding affinity for said lectin, such lectin being a three dimensional chemical structure other than a polynucleotide that binds to said nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by said lectin.  
     
     
         17 . The nucleic acid ligand of  claim 15  wherein said lectin is selected from the group consisting of a mammalian lectin, a plant lectin, a microbial lectin and a viral lectin.  
     
     
         18 . The nucleic acid ligand of  claim 15  wherein said lectin is selected from the group consisting of wheat germ agglutinin, L-selectin, E-selectin and P-selectin.  
     
     
         19 . The nucleic acid ligand of  claim 15  wherein said lectin is wheat germ agglutinin.  
     
     
         20 . The nucleic acid ligand to wheat germ agglutinin of  claim 19  wherein said nucleic acid ligand is a ribonucleic acid (RNA) ligand.  
     
     
         21 . The nucleic acid ligand of  claim 20  which comprises a modified ribonucleic acid.  
     
     
         22 . The nucleic acid ligand of  claim 21  wherein said modified ribonucleic acid is a 2′-amino (NH 2 ) modified ribonucleic acid.  
     
     
         23 . The nucleic acid ligand to wheat germ agglutinin of  claim 22  wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 2.  
     
     
         24 . The nucleic acid ligand of  claim 23  wherein said ligand is selected from the group consisting of SEQ ID NOS: 455.  
     
     
         25 . The nucleic acid ligand of  claim 20  wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS: 5-63.  
     
     
         26 . The nucleic acid ligand to wheat germ agglutinin of  claim 19  wherein said ligand is substantially homologous to and has substantially the same ability to bind said wheat germ agglutinin as a ligand selected from the group consisting of the sequences set forth in Table 2.  
     
     
         27 . The nucleic acid ligand to wheat germ agglutinin of  claim 19  wherein said ligand has substantially the same structure and the same ability to bind said wheat germ agglutinin as a ligand selected from the group consisting of the sequences set forth in Table 2.  
     
     
         28 . The nucleic acid ligand of  claim 15  wherein said lectin is a selectin.  
     
     
         29 . The nucleic acid ligand of  claim 28  wherein said selectin is selected from the group consisting of L-selectin, E-selectin and P-selectin.  
     
     
         30 . The nucleic acid ligand of  claim 29  wherein said selectin is L-selectin.  
     
     
         31 . The nucleic acid ligand to L-selectin of  claim 30  wherein said nucleic acid ligand is ribonucleic acid (RNA) ligand.  
     
     
         32 . The nucleic acid ligand of  claim 31  which comprises a modified ribonucleic acid.  
     
     
         33 . The nucleic acid ligand of  claim 32  wherein said modified ribonucleic acid is selected from the group consisting of a 2′-amino (2′-NH 2 ) modified ribonucleic acid and a 2′-fluoro (2′-F) modified ribonucleic acid.  
     
     
         34 . The nucleic acid ligand to L-selectin of  claim 33  wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Tables 8 and 16.  
     
     
         35 . The nucleic acid ligand of  claim 34  wherein said ligand is selected from the group consisting of SEQ ID NOS: 67-117 and 293-388.  
     
     
         36 . The nucleic acid ligand of  claim 31  wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS: 118-125.  
     
     
         37 . The nucleic acid ligand to L-selectin of  claim 30  wherein said ligand is substantially homologous to and has substantially the same ability to bind said L-selectin as a ligand selected from the group consisting of the sequences set forth in Tables 8, 12 and 16.  
     
     
         38 . The nucleic acid ligand to L-selectin of  claim 30  wherein said ligand has substantially the same structure and the same ability to bind said L-selectin as a ligand selected from the group consisting of the sequences set forth in Tables 8, 12 and 16.  
     
     
         39 . The nucleic acid ligand to L-selectin of  claim 30  wherein said nucleic acid ligand is deoxyribonucleic acid (DNA).  
     
     
         40 . The nucleic acid ligand to L-selectin of  claim 39  wherein said ligand is an DNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 12.  
     
     
         41 . The nucleic acid ligand of  claim 40  wherein said ligand is selected from the group consisting of SEQ ID NOS: 129-180 and 185-196.  
     
     
         42 . The nucleic acid ligand of  claim 39  wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS: 181-184.  
     
     
         43 . The nucleic acid ligand of  claim 29  wherein said selectin is P-selectin.  
     
     
         44 . The nucleic acid ligand to P-selectin of  claim 43  wherein said nucleic acid ligand is ribonucleic acid (RNA) ligand.  
     
     
         45 . The nucleic acid ligand of  claim 44  which comprises a modified ribonucleic acid.  
     
     
         46 . The nucleic acid ligand of  claim 45  wherein said modified ribonucleic acid is selected from the group consisting of a 2′-amino (2′-NH 2 ) modified ribonucleic acid, a 2′-fluoro (2′-F) modified ribonucleic acid, and a 2′-O-Methyl (2′-O-Me) modified ribonucleic acid.  
     
     
         47 . The nucleic acid ligand to P-selectin of  claim 46  wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Tables 19, 21 and 25.  
     
     
         48 . The nucleic acid ligand of  claim 47  wherein said ligand is selected from the group consisting of SEQ ID NOS: 199-219 and 236-290.  
     
     
         49 . The nucleic acid ligand of  claim 44  wherein said ligand comprises sequences selected from the group consisting of SEQ ID NO: 291.  
     
     
         50 . The nucleic acid ligand to P-selectin of  claim 43  wherein said ligand is substantially homologous to and has substantially the same ability to bind said P-selection as a ligand selected from the group consisting of the sequences set forth in Tables 19, 21 and 25.  
     
     
         51 . The nucleic acid ligand to P-selectin of  claim 43  wherein said ligand has substantially the same structure and the same ability to bind said P-selectin as a ligand selected from the group consisting of the sequences set forth in Tables 19, 21 and 25.  
     
     
         52 . The nucleic acid ligand to P-selectin of  claim 46  wherein said nucleic acid ligand is deoxyribonucleic acid (DNA).  
     
     
         53 . The nucleic acid ligand of  claim 15  wherein said ligand is a ribonucleic acid ligand.  
     
     
         54 . The nucleic acid ligand of  claim 53  which comprises a modified ribonucleic acid.  
     
     
         55 . The nucleic acid ligand of  claim 54  wherein said modified ribonucleic acid is selected from the group consisting of 2′-amino (2′-NH 2 ) modified ribonucleic acids, 2′-fluoro (2′-F) modified ribonucleic acids and 2′-O-Methyl (2′-O-Me) modified ribonucleic acids.  
     
     
         56 . The nucleic acid ligand of  claim 15  wherein said ligand is a deoxyribonucleic acid.  
     
     
         57 . The nucleic acid ligand of  claim 15  wherein said ligand has been further chemically modified at the sugar and/or phosphate and/or base.  
     
     
         58 . A multivalent Complex comprising a plurality of ligands of  claim 15 .  
     
     
         59 . A nucleic acid ligand to a lectin identified according to the method comprising: 
 a) contacting a candidate mixture of nucleic acids with a lectin, wherein nucleic acids having an increased affinity to said lectin relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;    b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and    c) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said lectin, whereby nucleic acid ligands of said lectin may be identified.    
     
     
         60 . A method for treating a lectin-mediated disease comprising administering a pharmaceutically effective amount of a nucleic acid ligand to a lectin.  
     
     
         61 . The method of  claim 60  wherein said nucleic acid ligand to a lectin is identified according to the method of  claim 1 .  
     
     
         62 . The method of  claim 60  wherein said lectin is a selectin.  
     
     
         63 . The method of  claim 62  wherein said selectin is L-selectin.  
     
     
         64 . The method of  claim 62  wherein said selectin is P-selectin.