Bacteria-Free Approach for Expressing RNA and Proteins in Human Cells

Technical Summary:

The Lucky7 approach combines two activities in a single 30-minute, room temperature reaction: the 5’-exonucleolytic activity of T7 DNA Exonuclease and the nick-sealing activity of T7 DNA ligase. In this reaction, precisely positioned phosphonothioate DNA modifications serve as stops for T7 DNA Exonuclease, producing fully defined perfectly matching single-stranded sticky ends for annealing of a DNA insert to nano-backbone. T7 DNA Ligase seals the newly formed insert-backbone nicks simultaneously with the process of sticky end formation and annealing. Following a 10- minute column clean-up, the reaction products are ready for use in human cells. In comparison to conventional E. coli-based DNA cloning methods, the Lucky7 approach requires none of the following procedures: DNA restriction digest, utilization of E. coli competent cells, transformation of DNA into E. coli, plating of transformed bacteria on LB plates followed by their overnight incubation, bacterial colony picking, secondary overnight growth of bacterial cultures in liquid LB media, bacterial culture centrifugation, mini/midi/maxipreps of plasmid from pelleted bacteria, and endotoxin removal from the bacteria derived plasmid DNA.