Figure 2.

Design of novel loop replacement library. (a) Library design consisted of introduction of a stop codon within the target loop sequence (1), semi-random motif design (2), an initial PCR round to amplify two overlapping fragments (3), and a final PCR to assemble the full-length cap gene containing the semi-random motif (4). (b) Four separate AAV2 libraries containing semi-random loops [L1, ~²⁶²SQSGASN~ (purple); L2, ~⁴⁴⁶SRTNTPSGTTTQSR~ (blue); L3, ~⁵⁴⁵QGSEKTNVDIEK~ (red); and L4, ~⁵⁸⁵RGNRQAATADVNT~ (green)] were constructed, with sequences determined through a bioinformatics analysis of AAV VP1 protein sequences. AAV, adeno-associated virus.