One-pot assembly of long, modified DNA via scaffolded oligo ligation

Background

As synthetic biology advances, the need for efficient construction of long, chemically modified DNA has become increasingly critical across applications such as gene editing, DNA data storage, epigenetics, and personalized therapeutics. These technologies require precise, site-specific incorporation of chemical tags into DNA strands that often span hundreds of nucleotides. However, conventional assembly methods such as PCR-based amplification or multi-step ligation often fail to retain chemical modifications and are prone to low yields, especially when repetitive or non-canonical sequences are involved. These constraints make it difficult to generate high-fidelity, full-length DNA products while preserving the structural and functional integrity of customized chemical features.

Technology overview

This DNA assembly method enables the synthesis of long, chemically modified DNA strands using a single-stranded DNA scaffold as a template. Short adapter oligonucleotides hybridize at defined intervals along the scaffold, forming docking sites for DNA fragments that connect adjacent adapters. Each fragment hybridizes with high specificity due to complementary domains that span between two adapters, ensuring that only correctly assembled products remain stable. A ligase then seals the fragments into a continuous DNA strand that replicates the scaffold sequence.

This one-pot, PCR-free process maintains all original chemical modifications, including methylation, fluorescent labels, and biotin. It also prevents misalignment of repetitive sequences and avoids intermediate fragment accumulation. By optimizing oligo ratios, the process yields high concentrations of full-length products in a single step. This approach offers improved fidelity, efficiency, and compatibility with complex sequence designs compared to conventional methods.