The　combination　of　chemotherapy　and　gene　therapy　holds　great　promise　for　the　treatment　and　eradication　of　tumors.　However,　due　to　significant　differences　in　physicochemical　properties　between　chemotherapeutic　agents　and　functional　nucleic　acid　drugs,　direct　integration　into　a　single　nano-agent　is　hindered,　impeding　the　design　and　construction　of　an　effective　co-delivery　nano-platform　for　synergistic　anti-tumor　treatments.　In　this　study,　we　have　developed　an　mRNA-responsive　two-in-one　nano-drug　for　effective　anti-tumor　therapy　by　the　direct　self-assembly　of　2′-fluoro-substituted　antisense　DNA　against　P-glycoprotein　(2′F-DNA)　and　chemo　drug　paclitaxel　(PTX).　The　2′-fluoro　modification　of　DNA　could　significantly　increase　the　interaction　between　the　therapeutic　nucleic　acid　and　the　chemotherapeutic　drug,　promoting　the　successful　formation　of　2′F-DNA/PTX　nanospheres　(2′F-DNA/PTX　NSs).　Due　to　the　one-step　self-assembly　process　without　additional　carrier　materials,　the　prepared　2′F-DNA/PTX　NSs　exhibited　considerable　loading　efficiency　and　bioavailability　of　PTX.　In　the　presence　of　endogenous　P-glycoprotein　mRNA,　the　2′F-DNA/PTX　NSs　were　disassembled.　The　released　2′F-DNA　could　down-regulate　the　expression　of　P-glycoprotein,　which　decreased　the　multidrug　resistance　of　tumor　cells　and　enhanced　the　chemotherapy　effect　caused　by　PTX.　In　this　way,　the　2′F-DNA/PTX　NSs　could　synergistically　induce　the　apoptosis　of　tumor　cells　and　realize　the　combined　anti-tumor　therapy.　This　strategy　might　provide　a　new　tool　to　explore　functional　intracellular　co-delivery　nano-systems　with　high　bioavailability　and　exhibit　potential　promising　in　the　applications　of　accurate　diagnosis　and　treatment　of　tumors.　©　2024　Elsevier　B.V.