TMGs were constructed as described previously (40 (link)). For non-synonymous point mutations, each mutated amino acid was flanked bilaterally by a sequence encoding 12 wild type (WT) amino acids to generate an individual minigene. For each frameshift mutation, a minigene was designed to contain preceding 12 WT amino acids followed by mutated amino acids in the new reading frame, which terminated at the new stop codon. Next, up to twelve minigenes were linked together to generate tandem minigenes, which were codon-optimized, synthesized and ligated into a pcDNA3.1 vector using an In-Fusion HD EcoDry Cloning Kit (Clontech/Takara, Mountain View, CA). TMG RNA was made by in vitro transcription using a HiScribe T7 Quick High Yield RNA Synthesis Kit (New England BioLabs, Ipswich, MA). RNA samples were purified using RNeasy Kit (Qiagen, Germantown, MD), quantified by spectrophotometry, and stored at −80°C until further use. The amino acid sequences of each TMG used in this study are shown in Supplementary Table 1.
Crude or HPLC-purified (>90% purity) 25-mer peptides, each encoding a point mutation flanked on both sides with 12 WT amino acids, were synthesized by GenScript (Piscataway, NJ) as lyophilized power, resuspended in DMSO and stored at −20°C until use.