A physical approach to prevention of secondary structure interference in oligonucleotide synthesis: memory-less elongation with a nanopore and sub-nm mechanical positioning

Synthetic oligonucleotide synthesis is hampered by secondary structures forming in the partially sequenced oligo and interfering with the elongation process. This limits the length of the oligo that can be synthesized and also makes it difficult to synthesize complex sequences; a number of chemical solutions with varying degrees of success are known. Here a physical solution is proposed at the single molecule level with an electrophoretic field and mechanical nanopositioning; it is similar to a recently proposed method for peptide synthesis without side-chain protection (doi: 10.26434/chemrxiv-2025-gsw8x-v5). Central to it are an electrolytic cell (e-cell) with a nanopore and a platform in the latter’s cis chamber that is moveable with sub-nanometer precision. Synthesis may be chemical or enzymatic, with an oligo synthesized as an extension to a header (initiator) DNA with one end immobilized on a glass slide mounted on the platform and the other threaded through the pore to expose the terminal nucleotide in the e-cell’s trans chamber. Synthesis, chemical or enzymatic, includes the following additional steps: 1) optional optical detection of elongation with Total Internal Fluorescence Reflection (TIRF) in enzymatic synthesis; 2) retraction of the elongated header-oligo into cis by approximately the length of a dNTP at the end of a cycle; and 3) full retraction/advance of the header-oligo into cis/trans on completion of synthesis for oligo separation. This is a memory-less Markov-like process in which the reagents in trans can only interact with the terminal nucleotide being extended and are oblivious of all previously added nucleotides, which are in the pore or cis and thus out of reach. As a result secondary structure and other issues arising from GC sequences, homonucleotide stretches, palindromes, etc. are no longer an issue; in principle there is no limit to the length of the oligonucleotide that can be synthesized in a single run. Header-pore arrays on a 5 cm chip with 1 micron pore separation (well above the resolution of currently available TIRF) can yield 2.5 billion oligonucleotides (~4 femtomoles); runtime is set by reaction rates and wash-and-refill steps in trans. Multiplexed synthesis can be done similarly but will require additional engineering of the platform and the e-cell.