A feasible one-pot approach for constructing oligonucleotide-grafted polymeric nanoparticles is reported. The approach involves formation of mesoglobules from a thermoresponsive polymer, coating of the mesoglobules with a cross-linked polymeric shell, and grafting the latter with oligonucleotide strands. Dynamic and static light scattering are used to parameterize the novel constructs. They are relatively large structures with hydrodynamic radii and molar masses reaching 200 nm and 150.0 × 106 g mol-1, respectively. The oligonucleotide-grafted polymeric nanoparticles are of spherical morphology and moderately negative (-12.4 to -19.1 mV) ζ potential as revealed by AFM, TEM, and electrophoretic light scattering. In accordance with their large size, they are found to carry thousands of oligonucleotide strands per particle. The novel constructs are thermoresponsive. They undergo reversible collapse upon heating and swelling upon cooling, which is associated with changes in the grafting density and, hence, the conformation of the oligonucleotide strands from unextended at room temperature to a more extended one at elevated temperatures. The versatility of the approach is demonstrated by varying the type of the cross-linked shell and content of the oligonucleotide strands and, hence, the grafting density. Appropriate diversification and modifications are suggested as well.