Random gene unidirectional antisense library

Abstract

The present invention provides a high-throughput system for functional genomics using a random gene unidirectional antisense library comprising LC-antisense compounds. The antisense compounds were specific and effective for the elimination of target mRNA. Thus, the system of the present invention may be effectively used as temporary knock-down system to unveil functions of genes critical for diseases. The system of the present invention can be adapted not only for functional genomics but also for effectively validating target for antisense or other molecular therapeutics against various malignancies, infections, and other diseases by blocking specific genes involved in the disease.

What is claimed is:  
     
         1 . A library of a multitude of single-stranded large circular nucleic acids, said library comprising: 
 a multiplicity of compartments, each of said compartments comprising one or more single-stranded large circular antisense molecule of bacteriophage or phagemid vector comprising at least one unidirectional antisense nucleic acid insert, 
 wherein said large circular antisense molecule is capable of being introduced into a host cell, and is capable of specifically binding to a nucleic acid in said host cell that is substantially complementary to said antisense nucleic acid insert.  
   
     
     
         2 . The library of  claim 1 , wherein the specificity of the antisense nucleic acid insert is unknown at the time said library is first made.  
     
     
         3 . The library of  claim 1 , wherein said host cell is a eucaryotic cell.  
     
     
         4 . The library of  claim 1 , wherein each of said compartments contains from about 0.1 μM to about 1 μM of said large circular antisense molecule.  
     
     
         5 . The library of  claim 1 , wherein said bacteriophage or phagemid vector is derived from a filamentous bacteriophage.  
     
     
         6 . The library of  claim 5 , wherein said filamentous bacteriophage is M13 bacteriophages.  
     
     
         7 . The library of  claim 1 , wherein the source of said nucleic acid insert is an eucaryotic organism.  
     
     
         8 . The library of  claim 1 , wherein said bacteriophage or phagemid vector comprises more than one kind of antisense nucleic acid insert sequence.  
     
     
         9 . The library according to  claim 1 , wherein said multiplicity of compartments comprises a multiwell format of at least 6 wells.  
     
     
         10 . The library according to  claim 1 , wherein said library is configured to be made and used in a substantially automated process.  
     
     
         11 . The library according to  claim 9 , wherein said multiplicity of compartments comprises a multiwell format of at least 96 wells.  
     
     
         12 . A method of making a library comprising a multitude of single-stranded large circular nucleic acids, which comprises one or more single-stranded bacteriophage or phagemid vector comprising at least one unidirectional antisense nucleic acid insert, comprising: 
 (i) inserting a nucleic acid fragment unidirectionally into said bacteriophage or phagemid vector by unidirectionally cloning the nucleic fragments into said vector;    (ii) preparing bacterial transformants by introducing the vector containing the insert into competent bacterial cells to make bacterial transformants; and    (iii) infecting said transformants with helper phage to produce said single-stranded nucleic acid library.    
     
     
         13 . A library of a multitude of single-stranded large circular nucleic acids, said library comprising: 
 a multiplicity of compartments, each of said compartments comprising one or more single-stranded large circular antisense molecule of bacteriophage or phagemid vector comprising at least one unidirectional subtracted antisense nucleic acid insert, 
 wherein said large circular antisense molecule is capable of being introduced into a host cell, and is capable of specifically binding to a nucleic acid in said host cell that is substantially complementary to said antisense nucleic acid insert.  
   
     
     
         14 . The library according to  claim 13 , wherein said unidirectional subtracted antisense nucleic acid is made by hybridizing a population of nucleic acids expressed from a first cell line or tissue with a population of nucleic acids expressed from a second cell line or tissue, and obtaining a nucleic acid population from the first cell line or tissue that does not hybridize with the nucleic acid population from said second cell line or tissue.  
     
     
         15 . The library according to  claim 14 , wherein said first cell line or tissue is abnormal such that modulation of gene expression is beneficial in returning said first cell line or tissue to normal, and wherein said second cell line or tissue is normal.  
     
     
         16 . The library according to  claim 15 , wherein said abnormality is cancer, viral infection, immunologic disorders or metabolic diseases.  
     
     
         17 . The library according to  claim 16 , wherein said cancer is liver cancer, lung cancer, stomach cancer, colon cancer, leukemia, thyroid cancer, skin cancer, prostate cancer, cervical cancer, or breast cancer.  
     
     
         18 . The library according to  claim 16 , wherein said viral infection is caused by human papilloma virus (HPV), HIV, small pox, mononucleosis (Epstein-Barr virus), hepatitis, or respiratory syncytial virus (RSV).  
     
     
         19 . The library according to  claim 16 , wherein said metabolic disease is phenylketonuria (PKU), primary hypothyroidism, galactosemia, abnormal hemoglobins, types I and II diabetes, or obesity.  
     
     
         20 . The library according to  claim 16 , wherein said immunological disorder is Sjogren's Syndrome, antiphospholipid syndrome, immune complex diseases, Purpura, Schoenlein-Henoch, immunologic deficiency syndromes, systemic lupus erythematosus, immunodeficiency, rheumatism, kidney, or liver sclerosis.  
     
     
         21 . A method of making a library comprising a multitude of single-stranded large circular nucleic acids, which comprises one or more single-stranded bacteriophage or phagemid vector comprising at least one unidirectional subtracted antisense nucleic acid insert, comprising: 
 (i) inserting a subtracted nucleic acid fragment unidirectionally into said bacteriophage or phagemid vector by unidirectionally cloning the subtracted nucleic fragments into said vector;    (ii) preparing bacterial transformants by introducing the vector containing the insert into competent bacterial cells to make bacterial transformants; and    (iii) infecting said transformants with helper phage to produce said single-stranded nucleic acid library.    
     
     
         22 . The method according to  claim 21 , wherein said subtracted nucleic fragment is made by hybridizing a population of nucleic acids expressed from a first cell line or tissue with a population of nucleic acids expressed from a second cell line or tissue, and obtaining a nucleic acid population from the first cell line or tissue that does not hybridize with the nucleic acid population from said second cell line or tissue.  
     
     
         23 . A method for specifically inhibiting growth of liver cancer cells, comprising administering to said cells large circular antisense molecules targeted to EST_Human IL3-UT0117-160301-504-H11; Apolipoprotein A-II, clone MGC:12334; PRO2675 mRNA; clone RP11-449G13 from 16; BAC clone RP11-360H4 from 2; gene supported by AK023036 (LOC90271); or gene similar to cytochrome b5 outer mitochondrial membrane precursor ( H. sapiens ) (LOC124229).  
     
     
         24 . A method for specifically inhibiting growth of liver cancer cells, comprising administering to said cells large circular antisense molecules targeted to HSPC025, clone MGC:4223 IMAGE:2959747; tissue inhibitor of metalloproteinase 1; alpha-fetoprotein (AFP); gene encoding protein FLJ14075; apolipoprotein A-II (APOA2); clone MGC:20176 IMAGE:3503710; eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2); cytochrome P450, subfamily IIE (ethanol-inducible) (CYP2E); or gene similar to serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1, clone MGC:9222 IMAGE:3859644.  
     
     
         25 . A high throughput system for functional genomics using a random gene unidirectional antisense library or random gene unidirectional subtracted antisense library comprising the following steps: 
 (i) forming large circular antisense molecule-carrier complexes with said unidirectional or unidirectional subtracted antisense libraries;    (ii) performing a primary gene functional analysis by transfecting the complexes into host cells to screen for the large circular antisense molecule that eliminates endogenously expressed substantially complementary transcripts;    (iii) identifying the large circular antisense molecule that eliminates the endogenously expressed transcript; and    (iv) sequencing either the antisense molecule or cDNA that corresponds to the antisense molecule.    
     
     
         26 . The high throughput system according to  claim 25 , further comprising, 
 (v) performing further gene function analysis with the large circular antisense molecule identified in steps (iii) and (iv).    
     
     
         27 . The high throughput system according to  claim 25 , comprising comparing the gene sequence obtained in step (iv) with a DNA sequence database to identify the gene.  
     
     
         28 . The high throughput system according to  claim 25 , wherein the carrier is liposomes, cationic polymers, HVJ-liposomes complexes, peptides or viruses.  
     
     
         29 . The high throughput system according to  claim 25 , wherein the large circular antisense molecule and carrier are mixed in an optimal ratio of about 1:3 to about 1:4 by weight.  
     
     
         30 . The high throughput system according to  claim 26 , wherein the gene function analysis is assaying for the phenotype of cell morphology, cell proliferation, cell apoptosis, or cell reaction to a substrate.  
     
     
         31 . The high throughtput system according to  claim 26 , wherein said gene function analysis is carried out by performing an assay, wherein said assay is RT-PCR, Western blot analysis, immunoassay, MTT reduction assay, [ 3 H]-thymidine incorporation assay, colony formation assay, DNA synthesis and chromatin activation, analysis of apoptosis by inspection of cell morphological changes, chromosomal condensation or fragmentation, DNA fragmentation, quantitative assay for apoptosis, signaling mechanisms of apoptosis, activation of cell cycle regulators, complex formation between cell cycle regulators, or assays for changes of metabolic, morphological, physiological and biochemical phenotypes in vitro and in vivo.