A Physics-Inspired Approach to Improve Oligonucleotide Design and Gene Synthesis.

Polymerase cycling assembly (PCA) allows gene synthesis from the assembly of overlapping oligonucleotides. For optimal yield, the sets of overlapping fragments should present uniform hybridization temperatures and minimal spurious dimer formation, conditions that become particularly difficult to fulfill for short, GC-biased, or dimer-prone sequences. We present a new algorithm for designing overlapping oligonucleotides for PCA. Unlike heuristic-based methods, the approach maps the design onto a statistical physics model whose low-temperature solution can be obtained exactly, through a transfer-matrix formalism, when spurious heterodimer interactions are negligible; when they are not, it is complemented by a simulated-annealing repair step and, optionally, by codon redesign, generating high-quality, thermodynamically consistent oligonucleotide sets. We evaluated the method on over 10,000 sequences previously deemed unsuitable for synthesis, successfully recovering roughly 80% of them, and more than 91% if codon redesign is allowed. Experimental validation on a subset of 14 representative designs confirmed the absence of dimer formation, with all assemblies yielding clean bands in agarose gel electrophoresis. Our approach offers a robust and reliable solution for oligonucleotide design, particularly in challenging sequence contexts, and represents a valuable tool for synthetic biology and automated gene assembly workflows.