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

• US 20090099043A1
• Filed: 11/21/2008
• Published: 04/16/2009
• Est. Priority Date: 07/23/2007
• Status: Abandoned Application

• Current Assignee: York YuanYuan Zhu
• Original Assignee: York YuanYuan Zhu
• Inventors: Zhu, York Yuan Yuan
• Application Number:
  US12/313,554
• Publication Number:
  US 20090099043A1
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2330/31 Libraries, arrays
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

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.

Claims

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.

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).

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.

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.