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201404 https://hdl.handle.net/2286/R.2.N.201404 http://rightsstatements.org/vocab/InC/1.0/ http://creativecommons.org/licenses/by-nc-sa/4.0 2025-05 49 pages Hassan, Youssef Hariadi, Rizal Van Horn, Wade Barrett, The Honors College School of Human Evolution & Social Change School of Molecular Sciences RNA interference (RNAi) utilizing small interfering RNA (siRNA) is a promising strategy for silencing the expression of harmful genes and proteins. However, its delivery, often mediated by endocytosis, can be inefficient, limiting the effectiveness of treatments. One of the current approaches to bypass the endocytic pathway typically involves cell-penetrating peptides (CPPs), but these CPPs are incapable of sequence-specific targeting. In this study, we propose a novel design using single-stranded (ssDNA), duplex (dsDNA), and hairpin DNA (hsDNA) anchored to a cholesterol moiety, enabling flip-flop-mediated translocation across the lipid bilayer. We find that ‘ssDNA flip’ across the membrane of giant unilamellar vesicles (GUVs) occurs with virtually complete efficiency. We also evaluated translocation efficiency with varied membrane composition and the position of cholesterol conjugation on ssDNA. Additionally, dsDNA displays delivery of extravesicular cargo into the GUVs via toehold-mediated strand displacement (TMSD). Finally, we engineered the flip-flop dynamics of hsDNA to result in signal transduction across GUVs and amplified the signal utilizing a hybridization chain reaction (HCR). Our approach provides a new perspective on the function of cholesterol-DNA conjugates as membrane-permeable systems for sequence-specific siRNA delivery as well as signal transduction and amplification platforms for diagnostic applications. DNA nanotechnology Cholesterol Signal Transduction DNA delivery Single Cholesterol Conjugated Flip-Flop Across the Lipid Bilayer