Construction of pool of interfering nucleic acids covering entire RNA target sequence and related compositions

Abstract

The present invention provides a PCR based high-throughput method for preparing full-sites siRNA polynucleotide pool, comprising: DNase I random digestion; Loop-1 phosphate linker ligation; single PCR amplification; a type III restriction/modification enzyme digestion; blunt ending; Loop-2 phosphate linker ligation; double primer PCR; FokI digestion and cloning into an siRNA expression vector. The present invention enables the use of a type III restriction/modification enzyme linkers mediated PCR method for high-throughput preparing an siRNA polynucleotide pool, in which the functional length of siRNAs can be controllably distributed from 19-23 bp, thus completely mimic the natural siRNA length diversity, specially suitable for RNAi therapeutic targets screening. The present invention overcomes the bottlenecks and drawbacks of conventional siRNA polynucleotide pool construction technologies.

1 . A method for producing a pool of interfering nucleic acid (iNA) polynucleotides comprising the following steps:
 a) partially digesting a DNA producing DNA constructs;   b) ligating Loop-1 linkers to one end of the DNA constructs wherein the loop-1 linker contains a type II restriction enzyme recognition site and a type III restriction enzyme recognition site;   c) cleaving the DNA constructs with a type III restriction/modification enzyme;   d) ligating a Loop-2 linker onto an end of each of the DNA constructs, wherein said Loop-2 linker has a type II restriction enzyme cognition site producing DNA constructs that have a Loop-1 linker at one end and a Loop-2 linker at a second end of the DNA construct;   e) digesting the DNA constructs with a type II restriction enzyme;   f) ligating the DNA constructs into iNA expression vectors; and   g) transfecting the transcription vectors into cells under conditions wherein iNAs are produced.   
     
     
         2 . The method of claim one wherein the RNA expression vectors have one or more RNA polymerase III promoters. 
     
     
         3 . The method of  claim 2  wherein the RNA expression vectors have two RNA polymerase III promoters, wherein a DNA construct that is to be transcribed into RNA is ligated into the vector between the two promoters. 
     
     
         4 . The method of  claim 3  wherein one of the promoters is a U6 promoter and one of the promoters is a H1 promoter. 
     
     
         5 . The method of  claim 1  wherein a termination signal is attached to each end of the DNA construct. 
     
     
         6 . The method of  claim 5  wherein the termination signals are polyA/polyT construct. 
     
     
         7 . The method of  claim 1  wherein the DNA construct of step (d) is amplified. 
     
     
         8 . The method of  claim 7  wherein the DNA construct is amplified by a polymerase chain reaction (PCR). 
     
     
         9 . A DNA construct for producing a iNA comprised of a first end, a second end and a middle DNA sequence in between the first in second ends wherein the first end is a loop-1 linker DNA sequence and the second end of the DNA construct is a loop-2 linker DNA sequence, wherein the middle DNA sequence encodes a iNA sequence having a length of from 16 to 27 bps, wherein the loop-1 linker has a type II restriction enzyme recognition site and a type III restriction enzyme site, and wherein the loop-2 linker DNA sequence has a type II restriction enzyme recognition site. 
     
     
         10 . The DNA construct of  claim 9  wherein at each end of the DNA construct is a termination signal for RNA polymerase III. 
     
     
         11 . The DNA construct of  claim 10  wherein the termination signal sequence is a polyA/polyT construct. 
     
     
         12 . The DNA construct of  claim 11  wherein the polyA sequence has 4 to 7 ‘A’s and the polyT has 4 to 7 ‘T’s. 
     
     
         13 . A iNA expression vector for producing a iNA comprised of two RNA polymerase III promoters and a DNA construct encoding an iNA, wherein the DNA construct has a first end and a second end, wherein the DNA construct is placed between the two promoters, wherein a RNA polymerase III termination signal sequence is ligated onto each end of the DNA construct and wherein the DNA is a sequence having 16 to 27 base-pairs of nucleotides. 
     
     
         14 . The expression vector of  claim 13  wherein the RNA polymerase III promoters are either a U6 RNA polymerase III promoter or a H1 RNA polymerase III promoter. 
     
     
         15 . The expression vector of construct of  claim 13  wherein the termination signal is a polyA/polyT construct.