Cell Membrane Targeting via Spacer Length-Engineered Amphiphilic DNA Frameworks

Cell membrane-anchored DNA nanostructures play a vital role in cellular sensing and engineering tools, yet their membrane-targeting efficiency is often limited by the mechanical mismatch between rigid frameworks and the highly dynamic lipid bilayer. Here, we systematically elucidate how spacer length-encoded structural flexibility governs the membrane-targeting performance of amphiphilic tetrahedral DNA frameworks (TDFs). By inserting poly(thymine) spacers of defined length between a rigid tetrahedral scaffold and cholesterol anchoring moieties, spacer length-encoded flexibility was investigated as a tunable structural feature without altering framework geometry or anchoring valency. Quantitative fluorescence analysis and live-cell confocal imaging reveal that spacer engineering markedly affects membrane targeting efficiency, with moderate increases in spacer length enhancing performance, whereas excessive extension may become unfavorable. Notably, TDFs bearing a 10-thymine spacer exhibit an approximately 6-fold increase in membrane-targeting signal compared with the spacer-free counterpart at short incubation times. Time-course analysis further reveals spacer-dependent differences in membrane targeting behavior, with the 10T construct showing the most rapid signal accumulation among the tested designs under the examined conditions. Together, these results identify spacer engineering as a key structural determinant for membrane targeting in amphiphilic DNA frameworks and provide a useful design strategy for improving the performance of DNA-based cell membrane probes.