A novel mild acetylation strategy for preparing high-resolution agarose gels targeting small-molecular-weight nucleic acid separation.

Small-molecular-weight nucleic acids (SMNs, 20-200 bp), such as microRNAs and short PCR amplicons, are crucial in molecular biology and diagnostics. Traditional agarose gels poorly resolve these fragments because of their large pore sizes, whereas high-resolution agarose (HRA) technologies are limited by harsh conditions, high costs, or narrow separation ranges. Here, a novel HRA was developed by acetylating three agaroses (Ag-1200, Ag-1500, and Ag-1800) with acetic anhydride, yielding optimal degrees of substitution (DS) of 0.32, 0.34, and 0.39, respectively. FTIR confirmed successful acetylation by the appearance of ester carbonyl groups at 1745 cm-1, and 13C NMR showed that acetylation primarily occurred at the C-2 hydroxyl group of β-D-galactose while preserving the agarose backbone. Small-angle X-ray scattering (SAXS) showed that the average characteristic structural size decreased from 10.6-11.0 nm to 8.7-9.4 nm, while Cryo-SEM revealed an increased proportion of pores in the 0-65 nm range. Gel electrophoresis demonstrated efficient separation of 20-200 bp SMNs, with 4% gels showing performance comparable to LONZA MetaPhor® Agarose and better than HyAgarose™ High Resolution Agarose. The modified agaroses also maintained gel strength above 460 g/cm2. This mild, cost-effective, and adaptable strategy provides a practical HRA alternative for SMN separation.