Inert splint-driven oligonucleotide assembly

In this study, we introduce a new in vitro method for oligonucleotide fragment assembly. Unlike polymerase chain assembly and ligase chain assembly that rely on short, highly purified oligonucleotides, our method, named Splynthesis, uses a one-tube, splint-driven assembly reaction. Splynthesis connects standard-desalted "contig" oligos (∼150 nt in length) via shorter "splint" oligos harboring 5' and 3' blocking modifications to prevent off-target ligation and amplification events. We demonstrate the Splynthesis method to assemble a 741-bp gene fragment. We verify the assembled polymerase chain reaction product using standard molecular biology techniques, as well as long-read Oxford Nanopore sequencing, and confirm that the product is cloneable via molecular means, as well as Sanger sequencing. This approach is applicable for synthetic biology, directed evolution, functional protein assays, and potentially even splint-based ligase chain reaction assays.