A DNA Double-Crossover Tile Overcomes the Length Limit of Template-Assisted DNA Cyclization through Distributed Multisite Hybridization

Abstract

Circular nucleic acids (circNAs) lack free termini, making them resistant to exonuclease degradation and valuable for applications in nucleic acid therapeutics, rolling circle amplification, DNA nanotechnology, and molecular diagnostics. Conventional template-assisted enzymatic and chemical cyclization methods efficiently generate relatively long circDNAs (>30 nt) but are less effective for shorter oligonucleotides because of the precursor’s insufficient stabilization while promoting its intermolecular ligation. Here, using 1-cyanoimidazole-mediated chemical ligation, we demonstrate that double-crossover (DX) DNA tiles overcome these limitations through a strategy of distributed multisite hybridization. By distributing hybridization interactions across the entire precursor and precisely positioning the reactive termini, the DX tile enables efficient synthesis of fully phosphodiester-linked circDNAs as short as 16 nt and consistently outperforms conventional ssDNA splint templates. We further show that precursors with both phosphorylated termini undergo efficient template-free cyclization through intramolecular phosphoanhydride bond formation, producing circDNA 14−50 nt in size with a 48−80% yield. Finally, we systematically characterized the electrophoretic behavior of circular and linear DNA homologs by identifying the electrophoretic mobility inversion pointthe oligonucleotide length at which circDNA changes from migrating faster than its linear counterpart to migrating slower. The inversion occurred at sizes 40, 30, and 22 nt in 8%, 12%, and 20% denaturing polyacrylamide gels, respectively, demonstrating that it is governed by gel composition. Together, these findings establish distributed multisite hybridization as an effective design principle for the synthesis of short circular DNA and provide new mechanistic insight into the electrophoretic behavior of circular nucleic acids.