The　combination　of　gene　therapy　and　chemotherapy　provides　a　We　developed　a　simple　and　versatile　approach　to　prepare　a　series　of　two-in-one　nanodrugs　through　direct　self-assembly　of　cyanine-labeled　single-stranded　DNA　(Cys-DNA)　and　different　types　of　drug　molecules.　Molecular　dynamics　simulation　showed　that　the　Cys　introduced　into　the　DNA　could　enhance　the　noncovalent　interaction　between　Cys-DNA　and　drug　molecules.　More　drug　molecules　were　incorporated　into　Cys-DNA,　tending　to　spontaneously　form　hybrid　Cys-DNA/drug　nanosphere.　Such　nanospheres　serve　as　both　carriers　and　cargoes,　excluding　the　extra　use　of　nontherapeutic　excipients　and　showing　ultrahigh　drug　loading　capacity.　Following　this　approach,　an　antisense　oligonucleotides/doxorubicin　nanodrug　model　was　constructed,　demonstrating　the　significant　synergistic　anti-tumor　therapeutic　effect.　As　a　proof　of　the　concept,　our　study　establishes　a　simple　and　reproducible　two-in-one　nucleic　acid-based　drug　formulation.　©　2021　Wiley-VCH　GmbH