Abstract: Short deoxyribonucleic acid molecules that are partially single-stranded, dumbbell-shaped, and covalently closed, which contain one or more unmethylated cytosine guanosine motif (CpG motif) and exhibit immunomodifying effects. Such molecules can be used for immunostimulation applications in humans or vertebrates.

Abstract: A process to obtain linear double-stranded covalently closed DNA “dumbbell” constructs from plasmids by restriction digest, subsequent ligation with hairpin oligodesoxyribonucleotides, optionally in the presence of restriction enzyme, and a final digestion with endo- and exonucleolytic enzymes that degrade all contaminating polymeric DNA molecules but the desired construct. The invention also provides a process to obtain said dumbbell constructs employing endonuclease class II enzymes. Furthermore, the invention provides a process to obtain linear, covalently closed DNA molecules, such as plasmids, free from contamination by genomic DNA, by submitting the DNA preparation to a facultative endonucleolytic degradation step and an obligatory exonucleolytic degradation step.