Nucleic acid molecule having binding affinity to a target molecule and a method for generating the same

Abstract

The present invention is related to a method for generating a nucleic acid molecule capable of binding to a target molecule comprising the following steps: a) providing a reference nucleic acid molecule, wherein the reference nucleic acid molecule is capable of binding to the target molecule and wherein the reference nucleic acid molecule comprises a sequence of nucleotides, wherein the sequence of nucleotides comprises n nucleotides; b) preparing a first level derivative of the reference nucleic acid molecule, wherein the first level derivative of the reference nucleic acid molecule differs from the reference nucleic acid molecule at one nucleotide position, wherein the first level derivative is prepared by replacing the ribonucleotide at the one nucleotide position by a 2′-deoxyribonucleotide in case the reference nucleic acid has a ribonucleotide at the nucleotide position and wherein the first level derivative is prepared by replacing the 2′-deoxyribonucleotide at the one nucleotide position by a ribonucleotide in case the reference nucleic acid has a 2′-deoxyribonucleotide at the nucleotide position and wherein the nucleotide position at which the replacement is made is the modified nucleotide position; and c) repeating step b) for each nucleotide position of the reference nucleic acid molecule, thus preparing a group of first level derivatives of the reference nucleic acid molecule, wherein the group of first level derivatives of the reference nucleic acid molecule consists of n first level derivatives, wherein each of the first level derivatives of the reference nucleic acid molecule differs from the reference nucleic acid molecule by a single nucleotide replacement and wherein each of the first level derivatives of the reference nucleic acid molecule has a single modified nucleotide position which is different from the single modified nucleotide of all of the single modified nucleotide positions of the other first level derivatives of the group of first level derivatives of the reference nucleic acid molecule.

1 .- 97 . (canceled) 
     
     
         98 . An L-nucleic acid molecule capable of binding to a target molecule by a mechanism other than base pairing obtainable by a method comprising the following steps:
 a) providing a reference L-nucleic acid molecule, wherein the reference L-nucleic acid molecule binds the target molecule, and wherein the reference L-nucleic acid molecule comprises a sequence of L-nucleotides, wherein the sequence of L-nucleotides comprises n L-nucleotides;   b) preparing a first level derivative of the reference L-nucleic acid molecule, wherein the first level derivative of the reference L-nucleic acid molecule differs from the reference L-nucleic acid molecule at one nucleotide position; wherein the first level derivative is prepared by replacing 2′-deoxyribonucleotide at the one nucleotide position by a ribonucleotide in case the reference L-nucleic acid molecule has a 2′-deoxyribonucleotide at the one nucleotide position; wherein the first level derivative is prepared by replacing ribonucleotide at the one nucleotide position by a 2′-deoxyribonucleotide in case the reference L-nucleic acid molecule has a ribonucleotide at the one nucleotide position; and wherein the nucleotide position at which the replacement is made is the modified nucleotide position;   c) repeating step b) for each nucleotide position of the reference L-nucleic acid molecule, thereby preparing a group of first level derivatives of the reference L-nucleic acid molecule, wherein the group of first level derivatives of the reference L-nucleic acid molecule consists of n first level derivatives, wherein each of the first level derivatives of the reference L-nucleic acid molecule differs from the reference L-nucleic acid molecule by a single nucleotide replacement and wherein each of the first level derivatives of the reference L-nucleic acid molecule has a single modified nucleotide position which is different from the single modified nucleotide of all of the single modified nucleotide positions of the other first level derivatives of the group of first level derivatives of the reference L-nucleic acid molecule;   d) determining a binding characteristic of each of the n first level derivatives of the reference L-nucleic acid molecule that binds the target molecule, wherein the binding characteristic comprises binding affinity of the first level derivative(s) of the reference L-nucleic acid molecule that binds the target molecule, wherein the binding affinity is expressed as KD value; and   e) identifying first level derivative(s) of the reference L-nucleic acid molecule that binds the target molecule, comprising binding affinity that exceeds binding affinity of the reference L-nucleic acid molecule that binds the target molecule, thereby obtaining L-nucleic acid molecules that bind(s) the target molecule by a mechanism other than base pairing.   
     
     
         99 . The L-nucleic acid molecule according to  claim 98 , wherein first level derivative(s) of the reference L-nucleic acid molecule that binds the target molecule, identified in step e) comprise a binding affinity that exceeds a predetermined threshold value. 
     
     
         100 . The L-nucleic acid molecule according to  claim 99 , wherein the predetermined threshold value is Y with Y being the quotient of(binding affinity of the reference L-nucleic acid molecule)/(binding affinity of a first level derivative) and wherein Y>1, Y≥2, Y≥5 or Y≥10. 
     
     
         101 . The L-nucleic acid molecule according to  claim 98 , wherein the L-nucleic acid molecule comprises at least one modification. 
     
     
         102 . The L-nucleic acid molecule according to  claim 101 , wherein excretion rate of the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification group from an organism is decreased compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides. 
     
     
         103 . The L-nucleic acid molecule according to  101 , wherein the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification has an increased retention time in an organism compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides. 
     
     
         104 . An L-nucleic acid molecule capable of binding to a target molecule, wherein the L-nucleic acid molecule has a binding affinity to the target molecule, wherein the binding affinity of the L-nucleic acid molecule to the target molecule is increased compared to the binding affinity of a reference L-nucleic acid molecule to the target molecule, wherein
 a) the L-nucleic acid molecule comprises a sequence of nucleotides and the reference L-nucleic acid molecule comprises a sequence of L-nucleotides, or   b) the L-nucleic acid molecule comprises a sequence of L-nucleotides and at least one modification group and the reference L-nucleic acid molecule comprises a sequence of L-nucleotides and the at least one modification group,
 wherein the sequence of L-nucleotides of the L-nucleic acid molecule and the sequence of L-nucleotides of the reference L-nucleic acid molecule are at least partially identical with respect to the nucleobase moiety of the L-nucleotides but differ with respect to the sugar moiety of the L-nucleotides, 
 wherein the sequence of L-nucleotides of the L-nucleic acid molecule consists of both L-ribonucleotides and 2′-L-deoxyribonucleotides and wherein the sequence of L-nucleotides of the reference L-nucleic acid molecule consists of either L-ribonucleotides or 2′-L-deoxyribonucleotides. 
   
     
     
         105 . The L-nucleic acid molecule according to  claim 104 , wherein the L-nucleic acid molecule and/or the reference L-nucleic acid molecule are antagonists of an activity mediated by the target molecule. 
     
     
         106 . The L-nucleic acid molecule according to  claim 104 , wherein excretion rate of the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification group from an organism is decreased compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides. 
     
     
         107 . The L-nucleic acid molecule according to  claim 104 , wherein the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification has an increased retention time in an organism compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides. 
     
     
         108 . The L-nucleic acid molecule according to  claim 104 , wherein the L-nucleic acid molecule comprises at least one modification. 
     
     
         109 . The L-nucleic acid molecule according to  claim 108 , wherein excretion rate of the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification group from an organism is decreased compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides. 
     
     
         110 . The L-nucleic acid molecule according to  108 , wherein the L-nucleic acid molecule comprising a sequence of L-nucleotides and at least one modification has an increased retention time in an organism compared to an L-nucleic acid molecule consisting of the sequence of L-nucleotides. 
     
     
         111 . The L-nucleic acid molecule according to  claim 104 , wherein the nucleic acid molecule comprises a method for the treatment and/or prevention of a disease. 
     
     
         112 . The nucleic acid molecule according to of  claim 104 , wherein the nucleic acid molecule comprises a method for the diagnosis of a disease. 
     
     
         113 . A pharmaceutical composition comprising the L-nucleic acid molecule according to  claim 104  and a pharmaceutically acceptable carrier. 
     
     
         114 . A method comprising administering to a subject in need of treatment the L-nucleic acid molecule according to  claim 104  and a pharmaceutically acceptable carrier. 
     
     
         115 . A method comprising exposing a sample of a subject suspected of comprising a condition to the L-nucleic acid molecule according to  claim 104  and determining whether a complex is formed with said L-nucleic acid molecule.