Method for screening nucleic acid catalysts

Abstract

Nucleic acid catalysts, method of screening for variants of nucleic acid catalysts, synthesis of ribozyme libraries and discovery of gene sequences are described.

1 . A method for identifying variants of a nucleic acid catalyst comprising the steps of: 
 a) selecting at least three positions within said nucleic acid catalyst to be varied with a predetermined group of different nucleotides;    b) synthesizing a first class of different pools of said nucleic acid catalyst, wherein the number of pools synthesized is equal to the number of nucleotides in the predetermined group of different nucleotides, wherein at least one of the positions to be varied in each pool comprises a defined nucleotide selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture of nucleotides selected from the predetermined group of different nucleotides;    c) testing the different pools of said nucleic acid catalyst under conditions suitable for said pools to show a desired attribute and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant in subsequent steps;    d) synthesizing a second class of different pools of nucleic acid catalyst, wherein at least one of the positions to be varied in each of the second class of different pools comprises a defined nucleotide selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture of nucleotides selected from the predetermined group of different nucleotides;    e) testing the second class of different pools of said nucleic acid catalyst under conditions suitable for showing (a) desired attribute and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant in subsequent steps; and    f) repeating the process similar to steps d and e until every position selected in said nucleic acid catalyst to be varied is made constant.    
     
     
         2 . A method for identifying novel nucleic acid molecules in a biological system, comprising the steps of: 
 a) synthesizing a pool of nucleic acid catalyst(s) having a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence;    b) testing the pools of nucleic acid catalyst under conditions suitable for showing a desired effect in said biological system and identifying the nucleic acid catalyst showing said desired effect;    c) using an oligonucleotide comprising the sequence of the substrate binding domain of the nucleic acid catalyst showing said desired effect as a probe to screening said biological system for nucleic acid molecules complementary to said probe; and    d) isolating and sequencing said complementary nucleic acid molecules.