Extensive libraries of gene truncation variants generated byin vitrotransposition

The detailed analysis of the impact of deletions on proteins or nucleic acids can reveal important functional regions and lead to variants with improved macromolecular properties. We present a method to generate large libraries of mutants with deletions of varying length that are randomly distributed throughout a given gene. This technique facilitates the identification of crucial sequence regions in nucleic acids or proteins. The approach utilizes in vitro transposition to generate 5΄ and 3΄ fragment sub-libraries of a given gene, which are then randomly recombined to yield a final library comprising both terminal and internal deletions. The method is easy to implement and can generate libraries in three to four days. We used this approach to produce a library of >9000 random deletion mutants of an artificial RNA ligase enzyme representing 32% of all possible deletions. The quality of the library was assessed by next-generation sequencing and detailed bioinformatics analysis. Finally, we subjected this library to in vitro selection and obtained fully functional variants with deletions of up to 18 amino acids of the parental enzyme that had been 95 amino acids in length.