Figure 6

Decoy sequence search technique developed as a suitability assessment for oligonucleotide mapping workflow. (A) Venn diagram of the shared and unique oligonucleotide fragments of a theoretical RNase T₁ digestion of BNT162b2 Original construct and its reverse-sequence construct. This is one example of a decoy construct. The theoretical digestion predicts 302 unique-sequence oligonucleotides (74%) for each and 78 shared oligonucleotides (26%). (B) Venn diagram of the shared-mass and unique-mass oligonucleotide fragments of a theoretical RNase T₁ digestion of BNT162b2 Original construct and of its reserve-sequence construct. The theoretical digestion predicts 147 unique-mass oligonucleotides for each. Of these, 145 oligonucleotides (99%) are shared. (C) Decoy construct identification overlay on the base peak ion chromatogram of the BNT162b2 Original RNase T₁ digest acquisition. Each colored bar marks an oligonucleotide feature identified by the BioPharma Finder automated software as originating from the RNase T₁ digest of a decoy construct. The decoy constructs are random sequences containing the nucleotide composition of BNT162b2 Original. The common sequence elution region contains shorter oligonucleotides, most of which are common to any decoy constructs as well as the true construct when subjected to RNaseT₁ digestion. The unique sequence elution region contains longer oligonucleotides, most of which are unique to the true construct. The true construct oligonucleotides are similar enough to decoy sequence oligonucleotides for the automated software to provide decoy oligonucleotide assignments. The identifications in the unique sequence elution region are not preferentially assigned to a single decoy construct, demonstrating that none of the decoy constructs are the true construct. (D) Target and decoy construct identification overlay on the base peak ion chromatogram of the BNT162b2 Original RNase T₁ digest acquisition. Each colored bar marks an oligonucleotide feature identified by the BioPharma Finder automated software as originating from the RNase T₁ digest of a decoy or target construct. Oligonucleotides in the unique sequence elution region are mostly identified (> 95%) as belonging to the BNT162b2 Original construct. This confirms the identity of the true construct and verifies the oligonucleotide mapping workflow’s ability to correctly identify oligonucleotides via LC-MS/MS.