Cell-free protein expression using rolling circle amplification product

Abstract

Methods for in vitro transcription and translation from an RCA product are provided. The methods comprise providing a double-stranded RCA product, wherein the double-stranded RCA product consists essentially of tandem repeats of a minimalistic expression sequence. The methods further comprise expressing a protein from the double-stranded RCA product in a cell-free expression system.

1 . A method for in vitro transcription comprising:
 generating a ribonucleic acid (RNA) in a cell-free transcription reaction from a double-stranded rolling circle amplification (RCA) product comprising tandem repeats of a deoxyribonucleic acid (DNA) sequence of interest,   wherein the DNA sequence of interest comprises a promoter sequence and an RNA coding region,   wherein the DNA sequence of interest comprises a transcription termination sequence, and   wherein the double-stranded RCA product is devoid of any extraneous sequences that are required for propagation of a plasmid in a host cell.   
     
     
         2 . The method of  claim 1 , wherein the promoter sequence is located upstream of the RNA coding region in the DNA sequence of interest. 
     
     
         3 . The method of  claim 1 , wherein the DNA sequence of interest further comprises an insulator sequence. 
     
     
         4 . The method of  claim 3 , wherein the transcription termination sequence is located at a position of about 14 base pairs to about 22 base pairs upstream of the promoter sequence in the double-stranded RCA product. 
     
     
         5 . The method of  claim 1 , wherein the DNA sequence of interest further comprises a purification tag sequence. 
     
     
         6 . The method of  claim 1 , wherein the DNA sequence of interest is a codon-optimized sequence for enhancing RNA stability. 
     
     
         7 . The method of  claim 1 , wherein the generated RNA comprises tandem repeats of cistronic RNA species, wherein each cistronic RNA species comprises an RNA sequence coded by the DNA sequence of interest. 
     
     
         8 . The method of  claim 1 , wherein the double-stranded RCA product comprises a thioated nucleotide. 
     
     
         9 . The method of  claim 1 , wherein the cell-free transcription reaction is performed using a prokaryotic cell extract, a eukaryotic cell extract, or a combination thereof. 
     
     
         10 . A method for in vitro transcription, comprising:
 generating a double-stranded rolling circle amplification (RCA) product via rolling circle amplification of a deoxyribonucleic acid (DNA) mini-circle comprising a DNA sequence of interest; and   generating a ribonucleic acid (RNA) in a cell-free transcription reaction from the double-stranded RCA product,   wherein the DNA sequence of interest comprises a promoter sequence and an RNA coding region,   wherein the DNA sequence of interest comprises a transcription termination sequence,   and   wherein the DNA mini-circle is devoid of any extraneous sequences that are required for propagation of a plasmid in a host cell.   
     
     
         11 . The method of  claim 10 , wherein the double-stranded RCA product is used in the cell-free transcription reaction without any further processing. 
     
     
         12 . The method of  claim 10 , wherein the double-stranded RCA product comprises a thioated nucleotide. 
     
     
         13 . The method of  claim 10 , wherein the cell-free transcription reaction is performed using a prokaryotic cell extract, a eukaryotic cell extract, or a combination thereof. 
     
     
         14 . The method of  claim 10 , wherein the DNA sequence of interest further comprises an insulator sequence. 
     
     
         15 . The method of  claim 10 , wherein the DNA sequence of interest further comprises a purification tag sequence. 
     
     
         16 . The method of  claim 10 , wherein the DNA sequence of interest is a codon-optimized sequence for enhancing RNA stability. 
     
     
         17 . The method of  claim 10 , wherein the generated RNA comprises tandem repeats of cistronic RNA species, wherein each cistronic RNA species comprises an RNA sequence coded by the DNA sequence of interest. 
     
     
         18 . The method of  claim 10 , wherein the rolling circle amplification is performed using a final concentration of deoxyribonucleoside triphosphates (dNTPs) in a range of 10 μM to 10 mM. 
     
     
         19 . The method of  claim 10 , wherein the rolling circle amplification is performed using a random primer mixture comprising a nucleotide analogue, and wherein the nucleotide analogue is an inosine, a Locked Nucleic Acid (LNA) nucleotide, a Peptide Nucleic Acid (PNA) nucleotide, a thioated nucleotide, 2-amino-deoxyadenosine, 2-thio-deoxythymidine, a polycation nucleotide, or a Zip Nucleic Acid (ZNA) polycation modified nucleotide. 
     
     
         20 . The method of  claim 19 , wherein the random primer mixture has a sequence+N+N(atN)(atN)(atN)*N.