The　physicochemical　differences　between　DNA　and　other　molecules　pose　a　challenge　to　the　construction　of　DNA-based　nanostructures.　Herein,　we　propose　a　straightforward　approach　for　preparing　multifunctional　DNA-based　nanospheres　through　direct　self-assembly　of　2′-fluoro-substituted　single-stranded　DNA　(2′F-DNA)　with　various　small　molecules.　Molecular　dynamics　simulation　revealed　that　2′F　substitution　in　DNA　can　cause　the　repulsion　of　adjacent　PO43-　group,　leading　to　local　stretching　of　the　DNA　structure.　Moreover,　2′F　substituent　induced　the　regular　polarization　of　H2O　nearby　F　to　form　the　hydration　layer,　which　interrupts　inherent　interactions　among　bases.　In　this　way,　the　bases　of　2′F-DNA　chain　have　fewer　constraints　and　more　flexibility　in　conformation,　facilitating　their　non-covalent　interactions　with　other　molecules　and　enhancing　the　self-assembly　capacity　of　2′F-DNA.　Consequently,　2′F-DNA　can　bind　to　more　molecules,　tending　to　spontaneously　form　hybrid　DNA　nanospheres.　Following　this　approach,　a　chemo-gene　therapy　2′F-DNA/doxorubicin　model　was　designed,　showing　the　significant　synergistic　anti-tumor　therapeutic　efficacy.　Taken　together,　this　study　provides　an　expandable　approach　for　constructing　engineering　hybrid　nanospheres　using　DNA　and　other　small　molecules,　which　holds　great　potential　for　further　biological　applications.　©　2023　Elsevier　B.V.