Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions.
We describe the synthesis of a novel type of mRNA template and its use in the preparation of mRNA-protein fusions. A light-induced psoralen crosslinking reaction was used to attach a puromycin-containing oligonucleotide to the 3'-end of an mRNA template. The photo-crosslinked template was found to undergo efficient mRNA-protein fusion formation in rabbit reticulocyte lysate. Fusion formation was subsequently tested with templates carrying puromycin linkers of different length and chemical composition. Short linkers with multiple triethyleneglycol phosphate building blocks allowed the most efficient fusion formation under a wide range of salt conditions. The present method simplifies the preparation of mRNA-protein fusions and thus significantly accelerates the in vitro protein evolution procedure which involves repetitive cycles of fusion production and selection.
Kurz
M
M
Phylos Inc., 128 Spring Street, Lexington, MA 02421, USA.
Gu
K
K
Lohse
P A
PA
D003432:Cross-Linking Reagents; D005363:Ficusin / Q000737:chemistry; D009842:Oligopeptides; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis; D011691:Puromycin; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D009842:Oligopeptides; D010455:Peptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011691:Puromycin; C085706:FLAG peptide; D005363:Ficusin