Transfection kinetics and structural promoters

Abstract

The invention features methods of analyzing the kinetics properties of transfection reactions. Also featured are methods for creating structural promoters which are effectively unregulated by enhancers and repressors. The structural promoters are significantly more active than the native promoter sequences upon which they are based.

1 . A method of expressing a protein, said method comprising the steps of:
 (i) annealing a linear single stranded oligonucleotide to a supercoiled DNA;   (ii) ligating the 3′ end of said oligonucleotide to the 5′ end of said linear single stranded oligonucleotide to form a circular padlock oligonucleotide;   (iii) transfecting the resulting supercoiled DNA into a cell; and   (iv) culturing said cell under conditions that permit expression of said protein.   
     
     
         2 . The method of  claim 1 , wherein said 3′ end and said 5′ end of said oligonucleotide has regions of complementarity to at least 5 nucleotides of said supercoiled DNA. 
     
     
         3 . The method of  claim 2 , wherein said regions of complementarity are continuous and are contained within a promoter region of said supercoiled DNA. 
     
     
         4 . The method of  claim 1 , wherein said oligonucleotide is annealed to the non-coding strand of said supercoiled DNA. 
     
     
         5 . The method of  claim 1 , wherein said oligonucleotide is an RNA oligonucleotide. 
     
     
         6 . A method of expressing a protein, said method comprising the steps of:
 (i) forming a forced open promoter complex in a supercoiled DNA comprising a nucleic acid sequence that encodes said protein; wherein the forced open promoter complex comprises a circular padlock oligonucleotide;   (ii) transfecting the resulting supercoiled DNA into a cell; and   (iii) culturing said cell under conditions that permit expression of said protein.   
     
     
         7 . The method of  claim 6 , wherein the circular padlock oligonucleotide is formed by annealing a linear oligonucleotide to said supercoiled DNA upstream of said nucleic acid sequence that encodes said protein and ligating the 3′ end of said oligonucleotide to the 5′ end of said oligonucleotide; 
     
     
         8 . The method of  claim 7 , wherein the 5′ end of said oligonucleotide anneals to an upstream end of a target sequence of said supercoiled DNA and the 3′ end of said oligonucleotide anneals to a downstream end of said target sequence, wherein said upstream and downstream ends of said target sequence comprise a contiguous sequence of said supercoiled DNA. 
     
     
         9 . The method of  claim 8 , wherein said target sequence is a promoter region of said supercoiled DNA. 
     
     
         10 . The method of  claim 7 , wherein said oligonucleotide comprises at least 30 nucleotides. 
     
     
         11 . The method of  claim 7 , wherein said oligonucleotide is annealed to the non-coding strand of said supercoiled DNA. 
     
     
         12 . The method of  claim 7 , wherein said oligonucleotide is an RNA oligonucleotide. 
     
     
         13 . A method of producing a gene silencing RNA, said method comprising the steps of:
 (i) annealing a single-stranded linear oligonucleotide to a supercoiled DNA comprising a nucleic acid sequence that encodes a gene silencing RNA;   (ii) ligating the 5′ end of said oligonucleotide to the 3′ end of said oligonucleotide to form a circular padlock oligonucleotide that is topologically linked to said supercoiled DNA;   (iii) transfecting the resulting supercoiled DNA into a cell; and   (iv) culturing said cell under conditions that promote the production of said gene silencing RNA.   
     
     
         14 . The method of  claim 13 , wherein said oligonucleotide anneals to said supercoiled DNA upstream of said nucleic acid sequence that encodes the gene silencing RNA. 
     
     
         15 . The method of  claim 13 , wherein the 5′ end of said oligonucleotide anneals to an upstream end of a target sequence of said supercoiled DNA and the 3′ end of said oligonucleotide anneals to a downstream end of said target sequence, wherein said upstream and downstream ends of said target sequence comprises a contiguous sequence of said supercoiled DNA. 
     
     
         16 . The method of  claim 15 , wherein said target sequence is a promoter region of said supercoiled DNA. 
     
     
         17 . The method of  claim 13 , wherein said oligonucleotide comprises at least 30 nucleotides. 
     
     
         18 . The method of  claim 13 , wherein said oligonucleotide is annealed to a non-coding region of the supercoiled DNA. 
     
     
         19 . The method of  claim 13 , wherein said oligonucleotide is an RNA oligonucleotide.